Compounds for use in synthesis of peptidomimetics

ABSTRACT

Synthesis of O-benzotriazole and O-imidazole synthons are described. Uses of synthons in synthesis of azapeptides and other peptidomimetics, azapeptides and other peptidomimetics synthesized from the synthons and uses of azapeptides and other peptidomimetics are also described.

This application is a continuation of U.S. patent application Ser. No.16/869,794, filed May 8, 2020, which claims the benefit of U.S.Provisional Application No. 62/845,617, filed May 9, 2019, thedisclosures of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention is directed to compounds for use in synthesis ofazapeptides and other aza-amino acid conjugates; synthesis ofazapeptides and other aza-amino acid conjugates, and uses of azapeptidesand other aza-amino acid conjugates in drug discovery, diagnosis,prevention, inhibition, and treatment of diseases.

BACKGROUND OF THE INVENTION

The in vitro and in vivo stability and in vitro and in vivo half-livesof peptides are limited, e.g., by their rate of hydrolysis and enzymaticdegradation.

Azapeptides are analogs of peptides. An azapeptide contains asubstituted semicarbazide instead of one or more of the amino acidresidue(s) of the parent peptide. In other words, one or more ofα-carbon atom(s) of the parent peptide are replaced with a nitrogen atomin the azapeptide. As compared to the parent peptides, azapeptides arehydrolysed and degraded by enzymes at a slower rate.

Aza-amino acids have not been ideal synthons for use in synthesis ofazapeptides and other aza-amino acid conjugates. As compared to theparent peptides, azapeptides contain a nitrogen atom instead of one ormore of α-carbon atom(s). Due, to the reduced reactivity of the carbonylmoiety in the aza-amino acid residue relative to a natural amino acidcounterpart, an aza-peptide bond is more stable under the effect ofpeptidases. Thus, azapeptides are hydrolysed and degraded by peptidasesat a slower rate and exhibit, e.g., an improved metabolic stability,than the parent peptides.

Nevertheless, the rate of formation of the aza-peptide bond is muchslower than that of a typical peptide bond. Consequently, there is agreater potential of formation of unwanted side products duringazapeptide synthesis with aza-amino acids than with conventional aminoacids. An additional obstacle in utilizing aza-amino acids in synthesesof azapeptides is the orthogonal functionalization of the two availablenitrogen atoms in the hydrazine system. For these and other reasons,syntheses of azapeptides with aza-amino acids and conventional couplingagents was challenging prior to the present invention.

There is a need for compounds which overcome the limitations ofconventional aza-amino acids and/or allow, e.g., for a faster and/orcheaper and/or more efficient synthesis of azapeptides and otheraza-amino acid conjugates.

SUMMARY OF THE INVENTION

It is an object of the invention to provide compounds for synthesis ofazapeptides and other aza-amino acid conjugates.

It is another object of the invention to provide azapeptides and otheraza-amino acid conjugates that are more stable and/or more efficaciousthan their parent peptides.

It is yet an additional object of the invention to provide azapeptidediagnostic and therapeutic agents.

In connection with the above objects and others, the invention isdirected to compounds of Formula (IA):

wherein A is N-phthalimidyl (NPhth) or NR₁R₂,

R₁ is H,

R₂ is tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc),2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz), or

R and R₁ or R₂ are connected and together form a side chain radical ofproline;

X is a heteroaryl; and

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, threonine, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, serine, and glutamine. The heteroaryl maybe unsubstituted or substituted with one or more of the following: ahalogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH,—COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., —CF₃, —CHF₂,—CH₂F, —CBr₃, —CHBr₂, —CH₂Br, —CCl₃, —CHCl₂, —CH₂Cl), —NH₂, or —NH₃. Incertain embodiments, the heteroaryl is substituted with —CF₃. The sidechain radicals may be unsubstituted or substituted with one or more ofthe following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,Phth, Boc, Fmoc, Ddz, etc.). In certain embodiments, R is selected fromthe group consisting of H, methyl, isopropyl, isobutyl, benzyl, and sidechain radicals of aspartic acid, histidine, glutamic acid, tryptophan,lysine, methionine, tyrosine, isoleucine (including, R-isoleucine,S-isoleucine and RS-isoleucine), arginine, asparagine, and glutamine. Incertain embodiments, the heteroaryl is an imidazolyl or abenzotriazolyl. Imidazolyl or benzotriazolyl may be unsubstituted orsubstituted with one or more of the following: a halogen (Cl, F, or Br),a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl (e.g., —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂,—CH₂Br, —CCl₃, —CHCl₂, —CH₂Cl), —NH₂, or —NH₃. In certain embodiments,imidazolyl or benzotriazolyl is substituted with —CF₃. Compounds ofFormula (IA) could be used as building blocks or synthons for synthesisof azapeptides and other peptidomimetics.

In certain embodiments, a compound of Formula (IA) is a compound inwhich

A is N-phthalimidyl or NR₁R₂,

R₁ is H,

R₂ is tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl;

X is imidazolyl or benzotriazolyl; and

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, threonine, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, and glutamine. Imidazolyl andbenzotriazolyl may be unsubstituted or substituted with one or more ofthe following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂, —CH₂Br, —CCl₃, —CHCl₂,—CH₂Cl), —NH₂, or —NH₃. In certain embodiments, the imidazolyl andbenzotriazolyl are substituted with —CF₃. The side chain radicals may beunsubstituted or substituted with one or more of the following: ahalogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH,—COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., achloromethyl, a fluromethyl, etc.) or a protecting group (e.g., Phth,Boc, Fmoc, Ddz, etc.). In certain embodiments, R is selected from thegroup consisting of H, methyl, isopropyl, isobutyl, benzyl, and sidechain radicals of aspartic acid, histidine, glutamic acid, tryptophan,lysine, methionine, tyrosine, isoleucine (including, R-isoleucine,S-isoleucine and RS-isoleucine), arginine, asparagine, and glutamine.

In certain embodiments, a compound of Formula (IA) is a compound inwhich

A is N-phthalimidyl or NR₁R₂,

R₁ is H,

R₂ is tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl;

X is imidazolyl substituted with —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂,—CH₂Br, —CCl₃, —CHCl₂, —CH₂Cl; and

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, threonine, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, and glutamine. The side chain radicalsmay be unsubstituted or substituted with one or more of the following: ahalogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH,—COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., achloromethyl, a fluromethyl, etc.) or a protecting group (e.g., Phth,Boc, Fmoc, Ddz, etc.). In certain embodiments, R is selected from thegroup consisting of H, methyl, isopropyl, isobutyl, benzyl, and sidechain radicals of aspartic acid, histidine, glutamic acid, tryptophan,lysine, methionine, tyrosine, isoleucine (including, R-isoleucine,S-isoleucine and RS-isoleucine), arginine, asparagine, and glutamine.

In certain embodiments, a compound of Formula (IA) is a compound inwhich

A is N-phthalimidyl or NR₁R₂,

R₁ is H,

R₂ is tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl;

X is benzotriazolyl substituted with —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂,—CH₂Br, —CCl₃, —CHCl₂, or —CH₂Cl; and

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, threonine, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, and glutamine. The side chain radicalsmay be unsubstituted or substituted with one or more of the following: ahalogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH,—COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., achloromethyl, a fluromethyl, etc.) or a protecting group (e.g., Phth,Boc, Fmoc, Ddz, etc.). In certain embodiments, R is selected from thegroup consisting of H, methyl, isopropyl, isobutyl, benzyl, and sidechain radicals of aspartic acid, histidine, glutamic acid, tryptophan,lysine, methionine, tyrosine, isoleucine (including, R-isoleucine,S-isoleucine and RS-isoleucine), arginine, asparagine, and glutamine.

In certain embodiments, a compound of Formula (IA) is a compound inwhich X is imidazolyl or benzotriazolyl, and

(i) A and R are connected and form a side chain of proline, or

(ii) A is hydrogen, or a protecting group comprising phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, and R is selected fromthe group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, cysteine, serine, threonine, and glutamine. The side chainradicals may be unsubstituted or substituted with one or more of thefollowing: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,Phth, Boc, Fmoc, Ddz, etc.).

In certain embodiments, a compound of Formula (IA) is a compound inwhich X is imidazolyl substituted with —CF₃, —CHF₂, —CH₂F, —CBr₃,—CHBr₂, —CH₂Br, —CCl₃, —CHCl₂, -or —CH₂Cl, and

(i) A and R are connected and form a side chain of proline, or

(ii) A is hydrogen, or a protecting group comprising phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, and R is selected fromthe group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, cysteine, serine, threonine, and glutamine. The side chainradicals may be unsubstituted or substituted with one or more of thefollowing: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,Phth, Boc, Fmoc, Ddz, etc.).

In certain embodiments, a compound of Formula (IA) is a compound inwhich X is benzotriazolyl substituted with —CF₃, —CHF₂, —CH₂F, —CBr₃,—CHBr₂, —CH₂Br, —CCl₃, —CHCl₂, or —CH₂Cl, and

(i) A and R are connected and form a side chain of proline, or

(ii) A is hydrogen, or a protecting group comprising phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, and R is selected fromthe group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, cysteine, serine, threonine, and glutamine. The side chainradicals may be unsubstituted or substituted with one or more of thefollowing: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,Phth, Boc, Fmoc, Ddz, etc.).

In certain embodiments, compound of Formula (IA) is a compound in whichR₁ and R are CH₂CH₂CH₂, and R₂ is tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, and X is imidazolyl orbenzotriazolyl.

In certain embodiments, compound of Formula (IA) is a compound in whichR₁ and R are CH₂CH₂CH₂, and R₂ is tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl, and X is imidazolyl orbenzotriazolyl.

The invention is also directed in part to compounds of Formula (IA):

wherein

X is imidazolyl or benzotriazolyl, and wherein

(i) A and R are connected and form a side chain of proline, or

(ii) A is hydrogen, or a protecting group comprising phthalimidyl,tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl; and R is selected fromthe group consisting of side chain radicals of aspartic acid,phenylalanine, alanine, histidine, glutamic acid, tryptophan, valine,leucine, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, glycine,asparagine, cysteine, serine, threonine, and glutamine.

Compounds of Formula (IA) could be used in drug discovery and/or asbuilding blocks or synthons for synthesis of azapeptides and otherpeptidomimetics.

The invention is also directed in part to compounds of Formula (IB):

wherein R₂ is a protecting group (e.g., tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl,2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl), and X is imidazolyl orbenzotriazolyl. Compounds of Formula (IB) could be used in drugdiscovery and/or as building blocks or synthons for synthesis ofazapeptides and other peptidomimetics.

In certain embodiments, the invention is directed to Phth-protectedcarbamoyl aza-imidazole derivatives and Phth-protected carbamoylaza-benzotriazole derivatives of unnatural amino acids, including, e.g.,aza-imidazole derivatives and Phth-protected carbamoyl aza-benzotriazolederivatives of β-amino acids (e.g., L-β-homotyrosine, β-alanine,L-β-homoasparagine, L-β-homoalanine, L-β-homophenylalanine,L-β-homoproline, L-β-holysine, L-β-homorarginine, L-β-proline, etc.),aliphatic amino acids (e.g., 6-aminohexanoic acid,2-amino-3-methoxybutanoic acid, 1-aminocyclopentane-1-carboxylic acid,2-(aminooxy)acetic acid, 6-aminohaxanoic acid,2-[2-(amino)-ethoxy]-ethoxy}acetic acid), β-cyclohexyl-L-alanine,6-aminohexanoic acid, L-α,β-diaminopropionic acid, L-propargylglycinel,L-α,β-diaminopropionic acid, α-aminoisobutyric acid,β-(2-pyridyl)-L-alanine, β-(3-pyridyl)-L-alanine,β-cyclopropyl-L-alanine, β-t-butyl-L-alanine,(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid,(allyloxycarbonyl)-L-α,β-diaminopropionic acid, D-α,β-diaminopropionicacid, L-α,β-diaminopropionic acid,(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyricacid, (N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid,L-α,γ-diaminobutyric acid, 4-fluoro-L-phenylglycine,5,5,5-trifluoro-DL-leucine, epsilon-aminohexanoic-OH,L-α-t-butylglycine, L-2-amino-3-(dimethylamino)propionic acid,L-2-aminocaproic acid, L-allylglycine, lysine azide,(Nδ-4-methyltrityl)-L-ornithine, Arg(Me)(Pbf)-OH, dimethyl-L-arginine(symmetrical and unsymmetrical), L-2-amino-3-guanidinopropionic acid,L-citrulline, ε-acetyl-L-lysine, Lys(ivDde)-OH, Lys(Me)2-OH.HCl,Lys(Me3)-OHchloride, α-methyl-DL-glutamic acid, γ-carboxy-L-glutamicacid γ,γ-di-t-butyl ester, (N-γ-ethyl)-L-glutamine, 2,6-diaminopimelicacid, Glu(OAll)-OH, L-cysteic acid, α-methyl-DL-methionine,DL-buthionine, L-cysteic acid, L-selenomethionine,S-[2-(4-pyridyl)ethyl]-L-cysteine, S-[2-(4-pyridyl)ethyl]-L-cysteine,S-diphenylmethyl-L-cysteine, S-trityl-L-homocysteine,S-trityl-L-penicillamine, (Se-p-methoxybenzyl)-L-selenocysteine,β-hydroxyphenylalanine, 2-cyano-L-phenylalanine, L-thyroxine,O-methyl-L-tyrosine, β-methyl-DL-phenylalanine, 2-cyano-L-phenylalanine,L-thyroxine, O-methyl-L-tyrosine, β-methyl-DL-phenylalanine,2-cyano-L-phenylalanine, 3,4-dichloro-L-phenylalanine,3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine,3,4-dihydroxy-phenylalanine, 3-amino-L-tyrosine, 3-chloro-L-tyrosine,3-fluoro-DL-tyrosine, 3-nitro-L-tyrosine, 4-amino-L-phenylalanine,4-aminomethyl-L-phenylalanine, 4-(phosphonomethyl)-phenylalanine,4-benzoyl-D-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine,4-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine,4-iodo-L-phenylalanine, DL-m-tyrosine, 2,6-dimethyl-tyrosine,L-homophenylalanine, O-methyl-L-tyrosine, Phe(4-guanidino)-OH,O-benzyl-L-phosphotyrosine, (2S,3R)-3-phenylpyrrolidine-2-carboxylicacid, (2S,4S)-4-phenyl-pyrrolidine-2-carboxylic acid,(2S,3aS,7aS)-Octahydro-1H-indole-2-carboxylic acid,(2S,3R)-3-phenylpyrrolidine-2-carboxylic acid,(2S,4R)-(−)-4-t-butoxypyrrolidine-2-carboxylic acid,trans-4-Fluoro-L-proline, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoicacid, 4-amino-3-hydroxybutanoic acid, L-α-methylserine,(2S,3S)-2-amino-3-methoxybutanoic acid, Thr(β-D-GlcNAc(Ac)3)-OH,O-benzyl-L-phosphoserine, O-benzyl-D-phosphothreonine,O-benzyl-L-phosphothreonine, 4-methyl-DL-tryptophan,6-fluoro-DL-tryptophan, 6-methyl-DL-tryptophan, DL-7-azatryptophan,(R)-7-Azatryptophan, 5-benzyloxy-DL-tryptophan, 5-bromo-DL-tryptophan,5-chloro-DL-tryptophan, 5-fluoro-DL-tryptophan, 5-hydroxy-L-tryptophan,5-methoxy-L-tryptophan, 6-chloro-L-tryptophan, 6-methyl-DL-tryptophan,7-methyl-DL-tryptophan, DL-7-azatryptophan, 5-azido-pentanoic acid,2-Amino-N-(3-azidopropyl)-3-mercaptopropionamide,2-Amino-N-(3-azidopropyl)-3-mercaptopropionamide, Azidohomoalanine,L-propargylglycine.DCHA, azidolysine, p-azidophenylalanine,Azidohomoalanine, D-propargylglycine, L-propargylglycine, azidolysine,Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine, 2-(7′-octenyl)alanine, 2-(4′-pentenyl) alanine, 2-(4′-pentenyl)glycine, 2-(7′-octenyl)alanine, [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid],L-glutamic acid-γ-[2-(1-sulfonyl-5-naphthyl)-aminoethylamide],N-ε-(5-carboxyfluorescein)-L-lysine,N-ε-(5/6-carboxyfluorescein)-L-lysine, N-ε-(4,4-dimethylazobenzene-4′carbonyl)-L-lysine, Nε-2,4-dinitrophenyl-L-lysine,N-ε-[(7-methoxycoumarin-4-yl)-acetyl-L-lysine, glycosylated amino acids(e.g., Ser(β-D-GlcNAc(Ac)3)-OH, Thr(β-D-GlcNAc(Ac)3)-OH),3-azabicyclo[3.1.0]hexane-2-carboxylic acid, 4-amino-(1-carboxymethyl)piperidine, 4-phenylpiperidine-4-carboxylic acid,Nα-methyl-N-im-trityl-L-histidine, Nα-methyl-O-benzyl-L-serinedicyclohexylammonium salt,Nalpha-methyl-Nomega-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine,Nalpha-methyl-L-leucine, Nalpha-methyl-L-norvaline,Nalpha-methyl-L-phenylalanine, Nalpha-methyl-N-im-trityl-L-histidine,Nalpha-methyl-O-t-butyl-L-serine, Nalpha-methylglycine,21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid,{2-[2-(amino)-ethoxy]-ethoxy}acetic acid, 6-Amino-4-oxahexanoic acid,5-Amino-3-Oxapentamoic Acid, NH-(PEG)10-CH2CH2COOH,NH-(PEG)12-CH2CH2COOH, 9-Amino-4; 7-Dioxanonanoic acid, 9-Amino-4;7-Dioxanonanoic acid, 12-amino-4,7,10-trioxadodecanoic acid,15-amino-4,7,10,13-tetraoxapentadecacanoic acid,18-amino-4,7,10,13,16-pentaoxaoctadecanoic acid,21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid,NH-(PEG)8-CH2CH2COOH, 11-amino-3,6,9-trioxaundecanoic acid,N-(Fmoc-8-amino-3,6-dioxa-octyl)succinamic acid, —N-ε-acetyl-L-lysine,L-citrulline, Arg(Me)(Pbf)-OH, N-ω,ω-dimethyl-L-arginine (assymetricaland symmetrical), Lys(Me)2-OH chloride, N-ε,ε-t-methyl-L-lysine,Lys(Me3)-OH chloride, O-benzyl-L-phosphoserine,O-benzyl-D-phosphothreonine, O-benzyl-L-phosphothreonine,O-benzyl-L-phosphotyrosine.

The invention is also directed to compounds of Formula (II):

wherein R is selected from the group consisting of side chain radicalsof aspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, threonine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, serine, and glutamine; and M is anoptional substituent selected from the group consisting of a halogen(Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH, —COH,methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., —CF₃, —CHF₂,—CH₂F, —CBr₃, —CHBr₂, —CH₂Br, —CCl₃, —CHCl₂, —CH₂Cl), —NH₂, or —NH₃. Incertain embodiments, M is —CF₃. The side chain radicals may beunsubstituted or substituted with one or more of the following: ahalogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH,—COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., achloromethyl, a fluromethyl, etc.) or a protecting group (e.g., Phth,Boc, Fmoc, Ddz, etc.). In certain embodiments, R is selected from thegroup consisting of H, methyl, isopropyl, isobutyl, benzyl, and sidechain radicals of aspartic acid, histidine, glutamic acid, tryptophan,lysine, methionine, tyrosine, isoleucine (including, R-isoleucine,S-isoleucine and RS-isoleucine), arginine, asparagine, and glutamine.Compounds of Formula (II) could be used in drug discovery and/or asbuilding blocks or synthons for synthesis of azapeptides and otherpeptidomimetics.

The invention is also directed to compounds of Formula (III):

wherein R is selected from the group consisting of side chain radicalsof aspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, threonine, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, serine, and glutamine. The side chainradicals may be unsubstituted or substituted with one or more of thefollowing: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,N-Phth, N-Boc, N-Fmoc, N-Ddz, etc.). In certain embodiments, R isselected from the group consisting of H, methyl, isopropyl, isobutyl,benzyl, and side chain radicals of aspartic acid, histidine, glutamicacid, tryptophan, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, asparagine, andglutamine. Compounds of Formula (III) could be used in drug discovery,diagnosis, prevention and treatment of diseases, or as building blocksor synthons for synthesis of azapeptides and other peptidomimetics.

The invention is also directed to compounds of Formula (IV):

wherein R is selected from the group consisting of side chain radicalsof aspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, threonine, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, serine, and glutamine. The side chainradicals may be unsubstituted or substituted with one or more of thefollowing: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.) or a protecting group (e.g.,N-Phth, N-Boc, N-Fmoc, N-Ddz, etc.). In certain embodiments, R isselected from the group consisting of H, methyl, isopropyl, isobutyl,benzyl, and side chain radicals of aspartic acid, histidine, glutamicacid, tryptophan, lysine, methionine, tyrosine, isoleucine (including,R-isoleucine, S-isoleucine and RS-isoleucine), arginine, asparagine, andglutamine. Compounds of Formula (IV) could be used in drug discovery,diagnosis, prevention and treatment of diseases, or as building blocksor synthons for synthesis of azapeptides, e.g., for use in drugdiscovery, diagnosis, prevention and treatment of diseases.

The invention is further directed to the use of compounds of Formulas(IA), (IB), (II), (III), and (IV) in the preparation of compounds ofFormula (V):

wherein

is at the N-terminus and/or the C-terminus and/or at or adjacent to acleavage or a hydrolysis site of the compound of Formula (V);

wherein B is selected from the group consisting of hydrogen, —NH₂,—NNH₂, —CONH₂, —COOR₃, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl,—OH, an amino acid, an aza amino acid, a 2 to 60-mer peptide, a 2 to60-mer aza peptide, a 2 to 60-mer azatide;

D is selected from the group consisting of —OR₄, —OH, —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₃ and R₄ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.);

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine. Theside chain radical of aspartic acid, phenylalanine, alanine, histidine,glutamic acid, tryptophan, valine, leucine, lysine, methionine,tyrosine, isoleucine (including, R-isoleucine, S-isoleucine andRS-isoleucine), arginine, glycine, asparagine, serine, threonine,cysteine and glutamine may be unsubstituted or substituted with one ormore of the following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g.,methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.) Compounds of Formula (V) areazapeptide analogues of compounds of Formula (VI):

wherein B is selected from the group consisting of hydrogen, —NH₂,—NNH₂, —CONH₂, —COOR₃, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl,—OH, an amino acid, an aza amino acid, a 2 to 60-mer peptide, a 2 to60-mer aza peptide, a 2 to 60-mer azatide;

D is selected from the group consisting of —OR₄, —OH, —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₃ and R₄ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.);

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine. Theside chain radical of aspartic acid, phenylalanine, alanine, histidine,glutamic acid, tryptophan, valine, leucine, lysine, methionine,tyrosine, isoleucine (including, R-isoleucine, S-isoleucine andRS-isoleucine), arginine, glycine, asparagine, serine, threonine,cysteine and glutamine may be unsubstituted or substituted with one ormore of the following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g.,methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.). In the preferred embodiments,compounds of Formula (V) are more resistant to hydrolysis and/orenzymatic degradation than compounds of Formula (VI). In some of thesepreferred embodiments, compounds of Formula (V) are more potent thancompounds of Formula (VI), e.g., due to a better fit into a biologicalreceptor. Compounds of Formula (V) could be used, e.g., in drugdiscovery, diagnosis, prevention and treatment of diseases.

The invention is further directed to the use of compounds of Formulas(IA), (IB), (II), (III), and (IV) in the preparation of compounds ofFormula (V):

wherein

is adjacent to the N-terminus and/or the C-terminus of the compound ofFormula (V);

wherein B is selected from the group consisting of hydrogen, —NH₂,—NNH₂, —CONH₂, —COOR₃, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl,—OH, an amino acid, an aza amino acid, a 2 to 60-mer peptide, a 2 to60-mer aza peptide, a 2 to 60-mer azatide;

D is selected from the group consisting of —OR₄, —OH, —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₃ and R₄ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.);

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine. Theside chain radical of aspartic acid, phenylalanine, alanine, histidine,glutamic acid, tryptophan, valine, leucine, lysine, methionine,tyrosine, isoleucine (including, R-isoleucine, S-isoleucine andRS-isoleucine), arginine, glycine, asparagine, serine, threonine,cysteine and glutamine may be unsubstituted or substituted with one ormore of the following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g.,methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.) Compounds of Formula (V) areazapeptide analogues of compounds of Formula (VI):

wherein B is selected from the group consisting of hydrogen, —NH₂,—NNH₂, —CONH₂, —COOR₃, —COOH, —COH, —COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl,—OH, an amino acid, an aza amino acid, a 2 to 60-mer peptide, a 2 to60-mer aza peptide, a 2 to 60-mer azatide;

D is selected from the group consisting of —OR₄, —OH, —NH₂, —NNH₂,—NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH, —COC₁-C₄ alkyl,—COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a 2 to 60-merpeptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₃ and R₄ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.);

R is selected from the group consisting of side chain radicals ofaspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, isoleucine(including, R-isoleucine, S-isoleucine and RS-isoleucine), arginine,glycine, asparagine, serine, threonine, cysteine, and glutamine. Theside chain radical of aspartic acid, phenylalanine, alanine, histidine,glutamic acid, tryptophan, valine, leucine, lysine, methionine,tyrosine, isoleucine (including, R-isoleucine, S-isoleucine andRS-isoleucine), arginine, glycine, asparagine, serine, threonine,cysteine and glutamine may be unsubstituted or substituted with one ormore of the following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g.,methyl), hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.). In the preferred embodiments,compounds of Formula (V) are more resistant to hydrolysis and/orenzymatic degradation than compounds of Formula (VI). In some of thesepreferred embodiments, compounds of Formula (V) are more potent thancompounds of Formula (VI), e.g., due to a better fit into a biologicalreceptor. Compounds of Formula (V) could be used, e.g., in drugdiscovery, diagnosis, prevention, inhibition, and treatment of diseases.

In certain embodiments, in compounds of Formula (V) and compounds ofFormula (VI), B of each compound is independently selected from thegroup consisting of hydrogen, —NH₂, —NNH₂, —CONH₂, —COOR₃, —COC₁-C₄alkyl, —COC₁-C₄ haloalkyl, —OH, an amino acid, an aza amino acid, a 2 to60-mer peptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

D of each compound is independently selected from the group consistingof —OR₄, —NH₂, —NNH₂, —NHCOCH₃, —NHCH₃, —N(CH₃)₂, —CONH₂, —COOH, —COH,—COC₁-C₄ alkyl, —COC₁-C₄ haloalkyl, an amino acid, an aza amino acid, a2 to 60-mer peptide, a 2 to 60-mer aza peptide, a 2 to 60-mer azatide;

R₃ and R₄ is each independently selected from the group consisting ofC₁-C₆ alkyl (e.g., methyl), methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl (e.g., a chloromethyl, a fluromethyl, etc.) or a protectinggroup (e.g., Phth, Boc, Fmoc, Ddz, etc.); and

R of each compound is independently selected from the group consistingof side chain radicals of aspartic acid, phenylalanine, alanine,histidine, glutamic acid, tryptophan, valine, leucine, lysine, serine,threoinine, methionine, tyrosine, isoleucine (including, R-isoleucine,S-isoleucine and RS-isoleucine), arginine, glycine, asparagine, andglutamine. The side chain radical of aspartic acid, phenylalanine,alanine, histidine, glutamic acid, tryptophan, valine, leucine, lysine,methionine, tyrosine, isoleucine (including, R-isoleucine, S-isoleucineand RS-isoleucine), arginine, glycine, asparagine, may be unsubstitutedor substituted with one or more of the following: a halogen (Cl, F, orBr), a C₁-C₆ alkyl (e.g., methyl), hydroxyl, —COOH, —COH, methoxyl,ethoxyl, propoxyl, a C₁-C₆ haloalkyl (e.g., a chloromethyl, afluromethyl, etc.) or a protecting group (e.g., Phth, Boc, Fmoc, Ddz,etc.).

The invention is further directed to the use of compounds of Formulas(IA), (IB), (II), (III), and (IV) in the preparation of azabradykinin,including, e.g., aza-7 bradykinin, aza-2,8 bradykinin, aza-2 bradykinin,and aza-8 bradykinin:

The invention is also directed in part to a method of preparing acompound of Formula (V), the method comprising a step of activating acompound of Formula (IA), Formula (IB), Formula (II), Formula (III) orFormula (IV) to form an activated compound of Formula (IA), Formula(IB), Formula (II), Formula (III), or Formula (IV), a step of couplingthe activated compound of Formula (IA), Formula (IB), Formula (II),Formula (III), or Formula (IV) with N-terminal of an amino acid,N-terminal of an aza-mino acid, provided that, if a side chain of theamino acid or aza-amino acid contains a group selected from amino,amide, guanidino N, carboxyl, sulfhydryl, carboxyl, hydroxyl, indole,imidazole phenol, the group is protected with a protecting groupselected from tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl(Fmoc), or 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz),phthalimide (Phth), carboxybenzyl (Cbz),2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf), trityl ortriphenylmethyl (Trt), t-butyl ester (OtBu), t-butyl ether (tBu),S-t-butyl ether (StBu), allyloxycarbonyl (Aloc), methoxytrimethylbenzenesulfonyl (Mtr), 4,4-dimethyloxybenzhydryl (Mbh),2,2,5,7,8-pentamethyl-chroman-6-sulfonyl chloride (Pmc),2,4,6-trimethoxybenzyl (Tmob), allylester (OAI), acetamidomethyl (Acm),and the like to form a protected compound of Formula (V), and a step ofdeprotecting the protected compound of Formula (V), e.g., withhydrazine, piperadine, TFA, acetic acid, thioanisole, EDT, anisole,etc., to form the compound of Formula (V).

In certain embodiments, compounds of Formula (IA), Formula (IB) andFormula (IV) are activated by iodomethane (MeI).

In certain embodiments, the compound of Formula (IA), Formula (IB) andFormula (III) are activated by DIPEA in acetonitrile.

The invention is also directed to a method of preparing an azapeptidecomprising a step of activating a compound according to Formula (I); anda step of coupling the activated compound with N-terminal of an aminoacid, N-terminal of an aza-mino acid; wherein the azapeptide is acompound of Formula (V). The compound of Formula (I) may beunsubstituted or substituted with one or more of the following: ahalogen, a C₁-C₆ alkyl, hydroxyl, —COOH, —COH, methoxyl, ethoxyl,propoxyl, or a C₁-C₆ haloalkyl. The

in the compound of Formula (V) may be at the N-terminus and/or theC-terminus of the compound of Formula (V), is adjacent to the N-terminusand/or the C-terminus of the compound of Formula (V) or hydrolysis siteof the compound of Formula (V). In some embodiments, the compound ofFormula (I) is activated by iodomethane, the coupling is in acetonitrileand comprises addition of DIPEA, and is during solid phase azapeptidesynthesis. In additional embodiments, the compound of Formula (I) isactivated by iodomethane, the coupling is in acetonitrile and comprisesaddition of DIPEA, and is during liquid phase azapeptide synthesis.

In the methods of the invention, the azapeptide is preferably preparedin a yield of at least about 40% (by weight) (e.g., from about 45% toabout 65%, from about 50% to about 65%, or from about 55% to about 65%,etc.). In certain embodiments, the yield is greater than about 45%,about 50%, about 55%, or about 60%. Thus, the yield may, e.g., be about50%, about 55%, about 60%, or about 65%. In certain embodiments, theazapeptide is a di-azapeptide and is synthesized in a yield from about80% to about 98%.

The invention is also directed in part to a method of preparing acompound of Formula (V), the method comprising a step of coupling acompound of Formula (IA), (IB), (II), (III), or (IV) with an aza-aminoacid to form a protected di-azatide, and a step of deprotecting theprotected di-azatide, e.g., with hydrazine, TFA, acetic acid,thioanisole, EDT, anisole, a mixture of any of the foregoing, or anotherde-protecting compound, to form a compound of Formula (V).

The invention is also directed in part to a method of preparing acompound of Formula (V), the method comprising a step of coupling afirst compound of Formula (IA), (IB), (II), (III), or (IV) with an aminoacid, a second compound of Formula (IA), (IB), (II), (III), or (IV), apeptide, or an azatide in acetonitrile at a temperature from 15° C. to35° C., wherein the coupling is for a time period from about 20 minutesto about 6 hours, from about 30 minutes to about 5 hours, from about 40minutes to about 4 hours, from about 50 minutes to about 3 hours, fromabout 50 minutes to about 2 hours. In some of these embodiments, thecoupling is for a time period from about 30 minutes to about 90 minutesat a temperature from about 18° C. to about 25° C. From about 1 to about3 equivalents of DIPEA and from about 1 equivalents to about 1.4equivalents (preferably, about 1.1 equivalent) of the amino acid, thesecond compound of Formula (IA), (IB), (II), (III), or (IV), thepeptide, or the azatide may be added to the acetonitrile for thecoupling reaction. In some embodiments, no additional reagents are addedduring the coupling step. In certain embodiments, a compound that isbeing coupled is an azapeptides with activated carbamoyl imidazolemoiety and about 1.5 eq of an amino acid and about 1.0 eq of DIPEA areused, and the coupling is at room temperature under nitrogen for about20 hours. In certain embodiments, a compound that is being coupled is anazatide with activated carbamoyl imidazole moiety and about 1.5 eqhydrazines and about 1.0 eq DIPEA are used, and the coupling is at about40° C. under nitrogen for about 20 hours. In certain embodiments, acompound that is being coupled is an azapeptides with carbamoylbenzotriazole (HBt) moiety and about 1.5 eq. of an amino acid and about2.0 eq. of DIPEA are used, and the coupling is at about 40° C. undernitrogen for about 20 hours. In certain embodiments, a compound that isbeing coupled is an azapeptide with carbamoyl 1-O-benzotriazole (HOBt)moiety and about 1.1 eq. of an amino acid and about 2.0 eq DIPEA areused, and the coupling is at about 25° C. under nitrogen for 1 hour.

The invention is further directed in part to a solution phase synthesisof the compounds of Formula (V), the solution phase synthesis comprisinga step of converting a compound of Formula (IA), (IB), (II), (III), or(IV) to an amide of the compound of Formula (IA), (IB), (II), (III), or(IV), a step of deprotecting the amide of the compound of Formula (IA),(IB), (II), (III), or (IV), a step of coupling the deprotected amide ofFormula (IA), (IB), (II), (III), or (IV) with an additional compound ofFormula (IA), (IB), (II), (III), or (IV), or a protected amino acid, ora protected aza-amino acid to form a protected azapeptide, and a step ofdeprotecting the protected azapeptide to provide a compound of Formula(V).

The invention is further directed in part to a solid phase synthesis ofthe compounds of Formula (V), the solid phase synthesis comprising astep of coupling a protected compound of Formula (IA), (IB), (II),(III), or (IV) to a support, a step of deprotecting the protectedcompound of Formula (IA), (IB), (II), (III), or (IV), a step of couplingthe deprotected compound of Formula (IA), (IB), (II), (III), or (IV) toan additional protected compound of Formula (IA), (IB), (II), (III), or(IV), an additional protected amino acid, or an additional protectedaza-amino acid to form a protected peptide, and a step of deprotectingand cleaving the protected peptide to provide a compound of Formula (V).

The invention is also directed in part to a process of preparing acompound of Formula (V) comprising a step of cleaving a peptide at itsN-terminus and/or C-terminus, and a step of coupling the cleaved peptidewith a compound of Formula (IA), (IB), (II), (III), or (IV) to form acompound of Formula (V). In certain embodiments, the compound of Formula(IA), (IB), (II), (III), or (IV) is activated prior to the coupling withthe cleaved peptide.

The invention is also directed in part to a process of preparing acompound of Formula (V) comprising a step of cleaving a peptide at itscleavage site to form two smaller peptides, a step of replacing the lastamino acid of at least one of the smaller peptides with an aza-aminoacid to form an azapeptide, and a step of conjugating the azapeptidewith the remaining smaller peptide to provide a compound of Formula (V).

The invention is also directed in part to a process of preparing acompound of Formula (V) comprising hydrolizing a peptide at its cleavagesite, and reacting the cleaved peptide with a compound of Formula (IA),(IB), (II), (III), or (IV) to provide a compound of Formula (V).

The invention is further directed in part to a method of azapeptidesynthesis comprising reacting a compound of Formula (IA), (IB), (II),(III), or (IV) with an aza-amino acid, an amino acid, or a peptide toform the azapeptide, wherein the azapeptide is a compound of formula(V).

The compounds of Formula (IA), (IB), (II), (III), or (IV) and process ofthe invention allow, e.g., for preparation of a compound of Formula (V)in yields (% by weight) of at least about 40% (e.g., from about 45% toabout 65%, from about 50% to about 65%, or from about 55% to about 65%,etc.). In certain embodiments, the yield is greater than about 45%,about 50%, about 55%, or about 60%. Thus, the yield may, e.g., be about50%, about 55%, about 60%, or about 65%. In certain embodiments, thecompound of Formula (V) is a di-azapeptide and is synthesized in a yieldfrom about 80% to about 98%.

Definitions

The term “about” in the present specification means a value within 15%(±15%) of the value recited immediately after the term “about,”including the value equal to the upper limit (i.e., +15%) and the valueequal to the lower limit (i.e., −15%) of this range. For example, thephrase “about 100” encompasses any numeric value that is between 85 and115, including 85 and 115.

An “azapeptide” means a peptide in which one or more α-carbon(s) arereplaced by nitrogen trivalent atom(s).

An “azatide” means a peptide in which all α-carbons are replaced bynitrogen trivalent atoms.

An “α-nitrogen” means a nitrogen atom bonded to a carbonyl group in anazapeptide or an azatide. The carbon atom next to the α-nitrogen iscalled the β-carbon.

An “aza-amino acid” is defined as an amino acid where the chiralα-carbon atom is replaced by a nitrogen atom.

An “azapeptide analogue” means a compound which differs from a peptidethat it is an analogue of in that one or more α-carbon atoms of thepeptide have been replaced by a nitrogen atom with or without additionalstructural modification(s) to the side chain(s) of the amino acidresidues of the peptide. The one or more α-carbon atoms that arereplaced may, e.g., be at the N-termini of the peptide (i.e., the firstresidue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide). Despite having a backbone different from the peptide, theazapeptide analogue preserves, extends and/or improves functionalactivity of the peptide. The azapeptide analogue is more resistant todegradation than the peptide and/or has an improved therapeutic activitythan the peptide and/or has an improved selectivity for a biologicalreceptor than the peptide and/or improved affinity to a biologicalreceptor and/or reversed activity at a biological receptor (agonisticactivity instead of antagonist activity or antagonistic activity insteadof agonistic activity).

The term “heteroaryl” includes all aryl compounds with atoms other thanC and H.

The term “protected” as it is used herein means that one or moregroup(s) (e.g., —OH) in an amino acid, an aza-amino acid, a peptide, anazapeptide, or a compound is protected with a protecting group (e.g.,Phth, Ddz, etc.). Unless otherwise indicated, the term “protectinggroup” or “protective group,” when used to refer to part of a moleculesubjected to a chemical reaction, means a chemical moiety that is notreactive under the conditions of that chemical reaction, and which maybe removed to provide a moiety that is reactive under those conditions.Protecting groups include, for example, nitrogen protecting groups andhydroxy-protecting groups. Examples of protective group include, e.g.,benzyl, diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl,ethoxycarbonyl, Phth, Ddz, as well as other protective groups known tothose skilled in the art.

A “side chain radical” of aspartic acid, phenylalanine, alanine,histidine, glutamic acid, tryptophan, valine, leucine, lysine,methionine, tyrosine, isoleucine (including, R-isoleucine, S-isoleucineand RS-isoleucine), arginine, glycine, asparagine, and glutamine havethe following structures:

A “side chain radical of proline” is a secondary amine, in that thealpha-amino group is attached directly to the main chain, making the αcarbon a direct substituent of the side chain:

Amino acids which can be used in the present invention are L- andD-amino acids.

Unless otherwise indicated, the terms “prevent,” “preventing” and“prevention” contemplate an action that occurs before a patient beginsto suffer from the symptoms of specified disease or disorder, whichinhibits or reduces the severity of the disease or disorder or of one ormore of its symptoms. The terms encompass prophylaxis.

The compounds of the invention can be used in the form ofpharmaceutically acceptable salts derived from inorganic or organicacids. For clarity, the term “pharmaceutically acceptable salt[s]” asused herein generally refers to salts prepared from pharmaceuticallyacceptable acids or bases including inorganic acids and bases andorganic acids and bases. Suitable pharmaceutically acceptable baseaddition salts include, e.g., metallic salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc or organic saltsmade from lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Suitable non-toxic acids include inorganic and organic acidssuch as acetic, alginic, anthranilic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic,galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific acidsinclude, e.g., hydrochloric, hydrobromic, phosphoric, sulfuric, andmethanesulfonic acids. Examples of specific salts include, e.g.,hydrochloride and mesylate salts. Others are well-known in the art. See,e.g., Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing,Easton Pa.: 1990) and Remington: The Science and Practice of Pharmacy,19th ed. (Mack Publishing, Easton Pa.: 1995). The preparation and use ofacid addition salts, carboxylate salts, amino acid addition salts, andzwitterion salts of compounds of the present invention may also beconsidered pharmaceutically acceptable if they are, within the scope ofsound medical judgment, suitable for use in contact with the tissues ofhumans and lower animals without undue toxicity, irritation, allergicresponse, and the like, are commensurate with a reasonable benefit/riskratio, and are effective for their intended use. Such salts may alsoinclude various solvates and hydrates of the compound of the presentinvention.

Certain compounds of the present invention may be isotopically labelled,e.g., with various isotopes of carbon, fluorine, or iodine, asapplicable when the compound in question contains at least one suchatom. In preferred embodiments, methods of diagnosis of the presentinvention comprise administration of such an isotopically labelledcompound.

Certain compounds of the present invention may exist as stereoisomerswherein, asymmetric or chiral centers are present. These stereoisomersare “R” or “S” depending on the configuration of substituents around thechiral carbon atom. The terms “R” and “S” used herein are configurationsas defined in IUPAC 1974 Recommendations for Section E, FundamentalStereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The inventioncontemplates various stereoisomers and mixtures thereof and these arespecifically included within the scope of this invention. Stereoisomersinclude enantiomers and diastereomers, and mixtures of enantiomers ordiastereomers. Individual stereoisomers of compounds of the inventionmay be prepared synthetically from commercially available startingmaterials which contain asymmetric or chiral centers or by preparationof racemic mixtures followed by resolution well known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and optional liberation of theoptically pure product from the auxiliary as described in Furniss,Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical OrganicChemistry”, 5th edition (1989), Longman Scientific & Technical, EssexCM20 2JE, England, or (2) direct separation of the mixture of opticalenantiomers on chiral chromatographic columns or (3) fractionalrecrystallization methods.

Certain compounds of the present invention may exist as cis- or transisomers, wherein substituents on a ring may attach in such a manner thatthey are on the same side of the ring (cis) relative to each other, oron opposite sides of the ring relative to each other (trans). Suchmethods are well known to those of ordinary skill in the art, and mayinclude separation of isomers by recrystallization or chromatography. Itshould be understood that the compounds of the invention may possesstautomeric forms, as well as geometric isomers, and that these alsoconstitute an aspect of the invention.

Unless otherwise indicated, a “diagnostically effective amount” of acompound is an amount sufficient to diagnose a disease or condition. Ingeneral, administration of a compound for diagnostic purposes does notcontinue for as long as a therapeutic use of a compound, and could beadministered only once if such is sufficient to produce the diagnosis.

The term “Phth-protected carbamoyl aza-imidazole derivative of anunnatural amino acid” as used herein means an unnatural aza-amino acidcovalently bound (conjugated) to phthalimidyl at its N-terminus and toimidazole at its C-terminus. The unnatural amino acid may be substitutedand unsubstituted.

The term “Phth-protected carbamoyl aza-benzotriazole derivative of anunnatural amino acid” as used herein means an unnatural aza-amino acidcovalently bound (conjugated) to phthalimidyl at its N-terminus and tobenzotriazole at its C-terminus. The unnatural amino acid may besubstituted and unsubstituted.

The term “solid-phase synthesis” means a method in which molecules(e.g., amino acids, aza-amino acids, etc.) are covalently bound on asolid support material and synthesised step-by-step in a single reactionvessel utilising selective protecting group chemistry. In this method,building blocks are typically protected at all reactive functionalgroups. The order of functional group reactions can be controlled by theorder of deprotection. For example, in an aza-peptide synthesis, anamino-protected amino acid or an amino-protected aza-amino acid is boundto a solid phase material (e.g., low cross-linked polystyrene beads),forming a covalent bond between the carbonyl group and the resin, e.g.,an amido or an ester bond. Then, the amino group is deprotected andreacted with the carbonyl group of the next amino-protected amino acidor amino-protected aza-amino acid. This cycle is repeated to form thedesired peptide or aza-peptide chain. After all reactions are complete,the synthesised peptide or aza-peptide is cleaved from the bead.

The terms “solution phase synthesis” and “liquid phase synthesis” meansa method in which molecules (e.g., amino acids, aza-amino acids, etc.)are synthesized in a solution without being covalently bound on a solidsupport material.

The term “synthon” means a building block.

The term “room temperature” means 20° C.

The term “ambient temperature” means 18-28° C.

The terms “parent peptide” and “corresponding peptide” mean a nativepeptide (i.e., natural or convention peptide) that differs from anazapeptide in that one or more of the amino residue(s) of the nativepeptide is (are) replaced by a semicarbazide or a substitutedsemicarbazide (i.e., one or more α-carbon(s) of the native peptide arereplaced by nitrogen trivalent atom(s)) in the azapeptide. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

The term “phthalimidyl” means:

The term “phthaloyl” means:

The abbreviation “N-Phth” means “N-phthalimidyl.”

The abbreviation “Boc” means “tert-butoxycarbonyl.”

The abbreviation “Fmoc” means “9-fluorenylmethoxycarbonyl.”

The abbreviation “Ddz” means“2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl.”

The abbreviation “HOBt” means “1-OH-Benzotriazole.”

The abbreviation “SPPS” means “Solid Phase Peptide Synthesis.”

The abbreviation “TCCA” means “trichloroisocyanuric acid.”

The abbreviation “TBACl” means “tetrabutyl ammonium chloride.”

The abbreviation “Phth” means “phthaloyl.”

The abbreviation “Cbz” means “carboxybenzyl.”

The abbreviation “Pbf” means“2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl.”

The abbreviation “Trt” means “trityl or triphenylmethyl.”

The abbreviation “OtBu” means “O-t-butyl.”

The abbreviation “tBu” means “t-butyl.”

The abbreviation “StBu” means S-t-butyl ether.

The abbreviation “Aloc” means “allyloxycarbonyl.”

The abbreviation “Mtr” means “methoxytrimethylbenzene sulfonyl.”

The abbreviation “Mbh” means “4,4-dimethyloxybenzhydryl.”

The abbreviation “Pmc” means “2,2,5,7,8-pentamethyl-chroman-6-sulfonylchloride.”

The abbreviation “Tmob” means 2,4,6-trimethoxybenzyl.

The abbreviation “OAP” means “allyl ester.”

The abbreviation “Acm” means “acetamidomethyl.”

The abbreviation “DEAD” means “Diethyl Azodicarboxylate” (IUPAC nameN-ethyl-N-propan-2-ylpropan-2-amine).

In peptide chemistry, “deprotection” refers to a process of removing theprotecting groups (e.g., phthaloyl, Boc, Cbz, Fmoc, etc) by a chemicalagent. For example, Boc protecting group could be removed under acidicconditions (e.g., 4M HCl, or neat trifluoroacetic acid TFA); Fmocprotecting group could be removed under basic conditions when pH ishigher than 12 (20% pipyridine/DMF or DCM); and Phthaloyl group can becleaved, e.g., under basic conditions or by the use of hydrazine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is HPLC of the intermediate (M=641) with side product (M=493)from SPPS of Example 15.

FIG. 2 is HPLC of crude 8AzaBK (26) from SPPS of Example 15.

FIG. 3 is HPLC of isolated 8Aza-BK (26) of Example 15.

DETAILED DESCRIPTION

A replacement of one or more α-carbon(s) with nitrogen in a peptideconverts the peptide to an “azapeptide”; and replacement of allα-carbon(s) with nitrogen(s) in a peptide converts the peptide to an“azatide.”

Azapeptides and azatides are peptidomimetics and are generally moreresistant to enzymatic hydrolysis than corresponding peptides. Theincrease in resistance to enzymatic degradation may lead to increasedmetabolic stability of the compounds and/or an improved receptor binding(e.g., an improved affinity to the receptor). Therefore, azapeptides andazatides are useful tools for drug design, applications in medicinalchemistry, and in diagnosis, prevention and treatment of diseases, andmay be used, e.g., instead of peptides, as azapeptide analogues(“peptidomimetics”).

Compounds of Formula (IA), (IB), (II), (III), and (IV) of the presentinvention could be used as “building blocks” or synthons for thesynthesis of azapeptides and other peptidomimetics and aza-amino acidconjugates, including compounds of Formula (V) in a solution phasesynthesis, a solid phase synthesis or a synthesis comprising both asolution phase synthesis and a solid phase synthesis.

Compounds of Formula (IA), (IB), (II), and (III)

Compounds of Formula (IA), (IB), (II), and (III) are stable at 37° C. inan aqueous medium (e.g., an aqueous solution) with a pH of about 7(e.g., distilled water) for at least 30 minutes, 60 minutes, 90 minutes,1 hour, 2 hours, 3 hours, 4 hours or 5 hours.

Compounds of Formula (IA), (IB), (II), and (III) may be used as buildingblocks or synthons for synthesis of azapeptides and otherpeptidomimetics and aza-amino acid conjugates. The resulting azapeptidesand peptidomimetics may have utility in drug discovery, diagnosis,prevention, inhibition, and treatment of diseases.

In certain embodiments, compounds of Formula (IA), (IB), (II), and (III)are selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein “PG” is H or aprotecting group (e.g., Phth, Boc, Fmoc, Ddz, etc.).

In certain embodiments, compounds of Formula (IA), (IB), (II), and (III)are selected from the group consisting ofN-(((1H-benzo[d][1,2,3]triazol-1-yl)oxy)carbonyl)-N-(1,3-dioxoisoindolin-2-yl)glycine(Phth-aza-aspartic acid-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl benzyl(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-phenylalanine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(1,3-dioxoisoindolin-2-yl)(methyl)carbamate(Phth-aza-alanine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl((1H-pyrrol-2-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-histidine-carbamoyl-O-benzotriazole),3-((((1H-benzo[d][1,2,3]triazol-1-yl)oxy)carbonyl)(1,3-dioxoisoindolin-2-yl)amino)propanoicacid (Phth-aza-glutamic acid-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl((1H-indol-3-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-tryptophan-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(1,3-dioxoisoindolin-2-yl)(isopropyl)carbamate(Phth-aza-valine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(1,3-dioxoisoindolin-2-yl)(isobutyl)carbamate(Phth-aza-leucine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(4-aminobutyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-lysine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(1,3-dioxoisoindolin-2-yl)(2-(methylthio)ethyl)carbamate(Phth-aza-cysteine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(1,3-dioxoisoindolin-2-yl)(4-hydroxybenzyl)carbamate(Phth-aza-tyrosine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-ylsec-butyl(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-leucine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(1,3-dioxoisoindolin-2-yl)(3-guanidinopropyl)carbamate(Phth-aza-arginine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl (1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-glycine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(2-amino-2-oxoethyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-asparagine-carbamoyl-O-benzotriazole),1H-benzo[d][1,2,3]triazol-1-yl(3-amino-3-oxopropyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-glytamine-carbamoyl-O-benzotriazole), and pharmaceuticallyacceptable salts thereof.

Compounds of Formula (IV)

Compounds of Formula (IV) are stable at 37° C. in an aqueous medium(e.g., an aqueous solution) with a pH of about 7 (e.g., distilled water)for at least 30 minutes, 60 minutes, 90 minutes, 1 hour, 2 hours, 3hours, 4 hours or 5 hours.

Compound of Formula (IV) may be used as building blocks or synthons forsynthesis of azapeptides and other peptidomimetics. The resultingazapeptides and peptidomimetics may have utility in drug discovery,diagnosis, prevention, inhibition, and treatment of diseases.

In certain embodiments, compounds of Formula (IV) are selected from thegroup consisting of:

and pharmaceutically acceptable salts thereof, wherein “PG” is H or aprotecting group (e.g., Phth, Boc, Fmoc, Ddz, etc.).

In certain embodiments, compounds of Formula (IV) are selected from thegroup consisting ofN-(((1H-imidazol-1-yl)oxy)carbonyl)-N-(1,3-dioxoisoindolin-2-yl)glycine(Phth-aza-aspartic acid-carbamoyl-O-imidazole),1H-imidazol-1-ylbenzyl(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-phenylalanine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)(methyl)carbamate(Phth-aza-alanine-carbamoyl-O-imidazole), 1H-imidazol-1-yl((1H-pyrrol-2-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-histidine-carbamoyl-O-imidazole),3-((((1H-imidazol-1-yl)oxy)carbonyl)(1,3-dioxoisoindolin-2-yl)amino)propanoicacid (Phth-aza-glutamic acid-carbamoyl-O-imidazole), 1H-imidazol-1-yl((1H-indol-3-yl)methyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-tryptophan-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)(isopropyl)carbamate(Phth-aza-valine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)(isobutyl)carbamate(Phth-aza-leucine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(4-aminobutyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-lysine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)(2-(methylthio)ethyl)carbamate(Phth-aza-cysteine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)(4-hydroxybenzyl)carbamate(Phth-aza-tyrosine-carbamoyl-O-imidazole), 1H-imidazol-1-ylsec-butyl(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-iso-leucine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)(3-guanidinopropyl)carbamate(Phth-aza-arginine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-glycine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(2-amino-2-oxoethyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-asparagine-carbamoyl-O-imidazole), 1H-imidazol-1-yl(3-amino-3-oxopropyl)(1,3-dioxoisoindolin-2-yl)carbamate(Phth-aza-glutamine-carbamoyl-O-imidazole), and pharmaceuticallyacceptable salts thereof.

Synthesis of Azapeptides and Azatides

Compounds of Formula (IA), (IB), (II), (III), and (IV) can be coupled ina linear, stepwise, chain-lengthening fashion to each other, aminoacids, aza-amino acids, peptides, azapeptides, and azatides by solutionor liquid phase, solid-phase and mixed solution/solid phase syntheticmethodologies to construct compounds of Formulas (V).

Compounds of Formula (IA), (IB), (II), (III), and (IV) can also be used,e.g., as sub-monomers to elongate and/or cap peptides and azapeptides.

For example, in certain embodiments, compounds of Formula (IA), (IB),(II), and (IV) may be activated by iodomethane, and the activatedcompound may be coupled, e.g., a protected or unprotected aza-aminoacid; a protected or unprotected peptide; a protected or unprotectedazapeptide; a protected or unprotected azatide; or a protected orunprotected compound of Formula (IA), Formula (IB) Formula (II), Formula(III), or Formula (IV); or a protected or unprotected hydrazine, byeither solution or liquid phase synthetic methodologies, e.g., to form acompound of Formula (V). The amino acid, the aza-amino acid, thepeptide, the azapeptide, compound of Formula (IA), (IB), (II), (III),and (IV) may each be unsubstituted or substituted with one or more ofthe following: a halogen (Cl, F, or Br), a C₁-C₆ alkyl (e.g., methyl),hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl(e.g., a chloromethyl, a fluromethyl, etc.). The coupling may, e.g., befor up to about 20 hours. In certain embodiments, the coupling may becompleted in about 30 minutes, about 40 minutes, about 50 minutes, about60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about100 minutes, about 110 minutes, or about 120 minutes.

The methods of the invention may be used to synthesize azapeptides andazatides from 2 to 200 mers in length, e.g., di-azatides, tri-azatides,tetra-azapeptides, penta-azapeptides, etc. In certain embodiments, thepeptide is 9 mers in length.

In certain embodiments, the method of preparing an azapeptide or anazatide comprises hydrolysing a peptide, e.g., a compound of Formula(VI) into fragments and reacting one or more fragments with a compoundof Formula (IA), (IB), (II), (III), or (VI).

In certain embodiments, the method of preparing an azapeptide or anazatide comprises cleaving a peptide, e.g., a compound of Formula (VI),into fragments and reacting one or more fragments with a compound ofFormula (IA), (IB), (II), (III), or (VI).

In certain embodiments, the method of preparing an azapeptide or anazatide comprises cleaving an end of a peptide, e.g., a compound ofFormula (VI), and reacting the cleaved peptide with a compound ofFormula (IA), (IB), (II), (III), or (VI).

In certain embodiments, the method of preparing an azapeptide or anazatide comprises reacting a compound of Formula (IA), (IB), (II),(III), or (VI) with a truncated peptide.

In certain embodiments, the method of preparing an azapeptide or anazatide comprises conjugating a compound of Formula (IA), (IB), (II),(III), or (VI) with a truncated peptide, e.g., a compound of Formula(VI).

In certain embodiments, a method of azapeptide or azatide synthesiscomprises reacting (i) a benzotriazole derivative of an aza-amino acidcomprising an aza-amino acid covalently bound (conjugated) to aprotecting group at its N-terminus and to benzotriazole at itsC-terminus with (ii) a peptide to form the azapeptide or azatide,wherein the benzotriazole derivative of the aza-amino acid azapeptide orazatide is a compound of Formula (IA), (IB), (II) or (III).

In certain embodiments, a method of azapeptide or azatide synthesiscomprises reacting (i) an imidazole derivative of an aza-amino acidcomprising an aza-amino acid covalently bound (conjugated) to aprotecting group at its N-terminus and to imidazole at its C-terminus,wherein the aza-amino acid is selected from the group consisting ofaza-glycine, aza-alanine, aza-valine, aza-leucine, aza-isoleucine,aza-proline, aza-phenylalanine, aza-tyrosine, aza-tryptophan,aza-aspartic acid, aza-glutamic acid, aza-aspargine, aza-glutamine,aza-histidine, aza-lysine, and aza-arginine with (ii) a hydrazide toform an azapeptide. In certain embodiments, the imidazole derivative isa compound of Formula (IV).

Uses of Compounds of Formula (V)

Compounds of Formula (V) are azapeptide analogues of compounds ofFormula (VI). In the preferred embodiments, compounds of Formula (V) aremore resistant to hydrolysis and/or enzymatic degradation than compoundsof Formula (VI).

Compounds of Formula (V) may be used to inhibit peptidases, both invitro and in vivo. The peptidase may, e.g., be an endopeptidase, anexopeptidase, an aspartic protease, a glutamic protease, an asparaginepeptide lyase, or a retroviral protease.

In some of these preferred embodiments, compounds of Formula (V) aremore potent than compounds of Formula (VI), e.g., due to a better fitinto a biological receptor. Compounds of Formula (V) could be used,e.g., in drug discovery, diagnosis, prevention, inhibition, andtreatment of diseases.

Compounds of Formulas (V) may each comprise from 2 to 200 carbonylgroup(s). For example, compounds of Formula (V) may each comprise 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 43,44, 56, or 166 carbonyl groups. In certain embodiments, compounds ofFormula (V) comprise from 2 to 60 carbonyl groups, from 2 to 50 carbonylgroups, from 2 to 40 carbonyl groups, from 2 to 30 carbonyl groups, from2 to 25 carbonyl groups, from 2 to 20 carbonyl groups, from 2 to 15carbonyl groups, from 2 to 12 carbonyl groups, from 2 to 10 carbonylgroups, from 2 to 9 carbonyl groups, from 3 to 40 carbonyl groups, from3 to 30 carbonyl groups, from 3 to 25 carbonyl groups, from 3 to 20carbonyl groups, from 3 to 15 carbonyl groups, from 3 to 12 carbonylgroups, from 3 to 10 carbonyl groups, or from 3 to 9 carbonyl groups.

In certain embodiments, compounds of Formula (V) comprise from 2 to 200carbonyl groups and at least one α-nitrogen covalently bound to at leastone of said carbonyl groups, and have a greater bioavailability (e.g.,oral, transdermal, and/or intranasal) than a peptide structurallydifferent from the compounds of Formula (V) only in that that thepeptide comprises α-carbon instead of said α-nitrogen. In certainembodiments, the α-nitrogen is at the N-termini or C-termini of thecompounds of Formula (V). In certain embodiments, the α-nitrogen is atthe N-termini and the C-termini of the compounds of Formula (V). Incertain embodiments, the α-nitrogen is not at the N-termini and not atthe C-termini of the compounds of Formula (V), rather it is at acleavage or hydrolysis site(s) of the peptide.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of therapeutic peptides.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of diagnostic peptides.

Compounds of Formula (V) may be used in drug discovery, diagnosis,prevention, inhibition, and treatment of diseases.

In certain embodiments, compounds of Formula (V) comprise a backbonecomprising from 2 to 200 carbonyl groups and α-nitrogen covalently boundto at least one of said carbonyl groups, and are therapeuticallyeffective for the treatment of a disorder in a subject, while a peptidestructurally different from the compounds of Formula (V) only in thatthat the peptide comprises α-carbon instead of said α-nitrogen is nottherapeutically effective for the treatment of the disorder. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

In certain embodiments, compounds of Formula (V) comprise from 2 to 200carbonyl groups and α-nitrogen covalently bound to at least one of saidcarbonyl groups, and have a therapeutic efficacy greater than a peptidestructurally different from the compounds of Formula (V) only in thatthe peptide comprises an α-carbon instead of said α-nitrogen. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

In certain embodiments, compounds of Formula (V) comprise from 2 to 200carbonyl groups and α-nitrogen covalently bound to at least one of saidcarbonyl groups, and have a longer duration of therapeutic activity thana peptide structurally different from the compounds of Formula (V) onlyin that that the peptide comprises α-carbon instead of said α-nitrogen.The replacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

In certain embodiments, compounds of Formula (V) comprise from 2 to 75carbonyl groups and at least one α-nitrogen covalently bound to at leastone of said carbonyl groups, and have an in vivo half-life greater thana peptide structurally different from the compounds of Formula (V) onlyin that said at least one α-nitrogen is replaced with α-carbon. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

In certain embodiments, compounds of Formula (V) comprise a backbonecomprising from 2 to 75 carbonyl groups, wherein at least two carbonylgroups are covalently bound to a trivalent nitrogen, and compounds ofFormula (V) have an in vivo half-life greater than a peptidestructurally different from the compounds of Formula (V) only in thatone or more alpha nitrogen(s) of the compounds of Formula (V) isreplaced with alpha carbon(s). The replacement may be, e.g., at theN-termini of the peptide (i.e., the first residue of the peptide), atthe second residue of the peptide, the C-termini of the peptide (i.e.,the last residue of the peptide), the residue covalently bound to theC-termini of the peptide, and/or at another residue of the peptide(e.g., at the site of hydrolysis of the peptide).

In certain embodiments, compounds of Formula (V) comprise at least oneaza-amino acid, and have an in vivo half-life greater than a peptidestructurally different from the compounds of Formula (V) only in thatthe aza-amino acid(s) is replaced with a corresponding amino acid. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

In certain embodiments, compounds of Formula (V) comprise from 2 to 200carbonyl groups and α-nitrogen covalently bound to at least one of saidcarbonyl groups, and are more resistant to protease degradation than apeptide structurally different from the compounds of Formula (V) only inthat that the peptide comprises α-carbon instead of said α-nitrogen. Thereplacement may be, e.g., at the N-termini of the peptide (i.e., thefirst residue of the peptide), at the second residue of the peptide, theC-termini of the peptide (i.e., the last residue of the peptide), theresidue covalently bound to the C-termini of the peptide, and/or atanother residue of the peptide (e.g., at the site of hydrolysis of thepeptide).

In certain embodiments, compounds of Formula (V) comprise from 2 to 200carbonyl groups and α-nitrogen covalently bound to at least one of saidcarbonyl groups, and have a greater affinity to a biological receptorthan a peptide structurally different from the compounds of Formula (V)only in that that the peptide comprises α-carbon instead of saidα-nitrogen. The replacement may be, e.g., at the N-termini of thepeptide (i.e., the first residue of the peptide), at the second residueof the peptide, the C-termini of the peptide (i.e., the last residue ofthe peptide), the residue covalently bound to the C-termini of thepeptide, and/or at another residue of the peptide (e.g., at the site ofhydrolysis of the peptide).

In certain embodiments, compounds of Formula (V) comprises from 2 to 60carbonyl groups.

In certain embodiments, compounds of Formula (V) are linear.

In certain embodiments, compounds of Formula (V) are cyclic.

In certain embodiments, compounds of Formula (V) are pegylated.

In certain embodiments, compounds of Formula (V) are conjugated to animmunoglobulin.

In certain embodiments, compounds of Formula (V) comprise α-nitrogen atthe N-terminus of its backbone.

In certain embodiments, compounds of Formula (V) comprise α-nitrogen atthe C-terminus of its backbone

In certain embodiments, compounds of Formula (V) comprise two carbonylgroups and two α-nitrogens.

In certain embodiments, compounds of Formula (V) comprise three carbonylgroups and one α-nitrogen.

In certain embodiments, compounds of Formula (V) comprise three carbonylgroups and two α-nitrogens.

In certain embodiments, compounds of Formula (V) comprise three carbonylgroups and three α-nitrogens.

In certain embodiments, compounds of Formula (V) comprise four carbonylgroups and one α-nitrogen.

In certain embodiments, compounds of Formula (V) comprise four carbonylgroups and two α-nitrogens.

In certain embodiments, compounds of Formula (V) comprise four carbonylgroups and three α-nitrogens.

In certain embodiments, compounds of Formula (V) comprise four carbonylgroups and four α-nitrogens.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 2 to 200 amino acid peptide comprising an amino acidselected from the group consisting of glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid,glutamic acid, aspargine, glutamine, histidine, lysine, and arginine;the aza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 2 to 200 amino acid peptide comprising an amino acidselected from the group consisting of glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid,glutamic acid, aspargine, glutamine, histidine, lysine, and arginine,wherein the analogue includes at least one corresponding aza-amino acidof the amino acid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 2 to 200 amino acid peptide, the 2 to 200 amino acidpeptide comprising amino acids selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,tyrosine, tryptophan, aspartic acid, glutamic acid, aspargine,glutamine, histidine, lysine, arginine, the analogue differing from theamino acid peptide in that that the aza-analogues comprise an aza-aminoacid instead of at least one of the amino acids, wherein theaza-analogues comprise aza-glycine instead of glycine, and/or theaza-analogues comprise aza-alanine instead of alanine, and/or theaza-analogues comprise aza-valine instead of valine, and/or theaza-analogues comprise aza-leucine instead of leucine, or/and theaza-analogues comprise aza-isoleucine instead of iso-leucine, and/or theaza-analogues comprise aza-proline instead of proline, and/or theaza-analogues comprise aza-phenylalanine instead of phenylalanine,or/and the aza-analogues comprise comprises aza-tyrosine instead oftyrosine, and/or the aza-analogues comprise aza-tryptophan instead oftryptophan, or/and the aza-analogues comprise aza-aspartic acid insteadof aspartic acid, and/or the aza-analogues comprise aza-glutamic acidinstead of glutamic acid, and/or the aza-analogues compriseaza-aspargine instead of aspargine, and/or the aza-analogues compriseaza-glutamine instead of glutamine, and/or the aza-analogues compriseaza-histidine instead of histadine, and/or the aza-analogues compriseaza-lysine instead of lysine, and/or the aza-analogues compriseaza-arginine instead of arginine.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a peptide comprising from 2 to 50 amino acids selected fromthe group consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, arginine, and at least 2,3, 4, 5, 6, 7, 8, 9 or 10 of the amino acids are replaced withcorresponding aza-amino acids. In some of these embodiments, thereplaced amino acid is the first amino acid of the peptide. In some ofthese embodiments, the replaced amino acid is the last amino acid of thepeptide. In some of these embodiments, the first and the last aminoacids of the peptide are both replaced with corresponding aza-aminoacids.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 10-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 9-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 8-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 7-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 6-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 5-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 5-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 4-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, compounds of Formula (V) are azapeptideanalogues of a 3-mer peptide comprising an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, aspargine, glutamine, histidine, lysine, and arginine; theaza-analogues differing from the amino acid peptide in that that theamino acid of the peptide is replaced with a corresponding aza-aminoacid.

In certain embodiments, the last amino acid of the peptide is selectedfrom the group consisting of aspartic acid, phenylalanine, and arginine.

In certain embodiment, the first amino acid of the peptide is selectedfrom the group consisting of tyrosine, phenylalanine, and arginine.

In certain embodiments, the first and the last amino acid of the peptideare the same.

In certain embodiments, the first and the last amino acids of thepeptide are different.

In certain embodiments, compounds of Formula (V) are not azatides.

In certain embodiments, compounds of Formula (V) comprise an amino acidselected from the group consisting of cysteine, methionine, serine andthreonine.

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-glycine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-alanine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-valine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-leucine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-isoleucine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-proline(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-phenylalanine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-tyrosine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-tryptophan(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-aspartic acid(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-glutamic acid(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-aspargine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-glutamine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-histidine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-lysine(s).

In certain embodiments, compounds of Formula (V) comprise at least one,at least two or at least three aza-arginine(s).

In certain embodiments, compounds of Formula (V) comprise aza-glycine,aza-alanine, aza-valine, aza-leucine, aza-isoleucine, aza-proline,aza-phenylalanine, aza-tyrosine, aza-tryptophan, aza-aspartic acid,aza-glutamic acid, aza-aspargine, aza-glutamine, aza-histidine,aza-lysine, or aza-arginine on their N-termini and/or C-termini.

In certain embodiments, compounds of Formula (V) comprise aza-glycine,aza-alanine, aza-valine, aza-leucine, aza-isoleucine, aza-proline,aza-phenylalanine, aza-tyrosine, aza-tryptophan, aza-aspartic acid,aza-glutamic acid, aza-aspargine, aza-glutamine, aza-histidine,aza-lysine, or aza-arginine on their N-termini and/or C-termini, and areaza-analogues of a therapeutic peptide, and have a greaterbioavailability (e.g., oral, transdermal, and/or intranasal) than thetherapeutic peptide (in its unaltered state).

In certain embodiments, compounds of Formula (V) comprise aza-glycine,aza-alanine, aza-valine, aza-leucine, aza-isoleucine, aza-proline,aza-phenylalanine, aza-tyrosine, aza-tryptophan, aza-aspartic acid,aza-glutamic acid, aza-aspargine, aza-glutamine, aza-histidine,aza-lysine, or aza-arginine on their N-termini and/or C-termini, and areaza-analogues of a therapeutic peptide, maintain the therapeuticefficacy of the therapeutic peptide and have an in vivo half-lifegreater than the in vivo half-life of the therapeutic peptide.

In certain embodiments, compounds of Formula (V) comprise aza-glycine,aza-alanine, aza-valine, aza-leucine, aza-isoleucine, aza-proline,aza-phenylalanine, aza-tyrosine, aza-tryptophan, aza-aspartic acid,aza-glutamic acid, aza-aspargine, aza-glutamine, aza-histidine,aza-lysine, or aza-arginine on their N-termini and/or C-termini, areaza-analogues of a therapeutic peptide and have a longer duration oftherapeutic activity than the therapeutic peptide.

In certain embodiments, compounds of Formula (V) comprise aza-glycine,aza-alanine, aza-valine, aza-leucine, aza-isoleucine, aza-proline,aza-phenylalanine, aza-tyrosine, aza-tryptophan, aza-aspartic acid,aza-glutamic acid, aza-aspargine, aza-glutamine, aza-histidine,aza-lysine, or aza-arginine on their N-termini and/or C-termini, areaza-analogues of a therapeutic peptide and are more resistant toprotease degradation than the therapeutic peptide.

In certain embodiments, compounds of Formula (V) comprise aza-glycine,aza-alanine, aza-valine, aza-leucine, aza-isoleucine, aza-proline,aza-phenylalanine, aza-tyrosine, aza-tryptophan, aza-aspartic acid,aza-glutamic acid, aza-aspargine, aza-glutamine, aza-histidine,aza-lysine, or aza-arginine on their N-termini and/or C-termini, areaza-analogues of a therapeutic peptide and have a greater affinity to abiological receptor than the therapeutic peptide.

Di-azatides

In certain embodiments, a compound of Formula (V) is a di-azatide of acompound of Formula (IX)

or a pharmaceutically acceptable salt thereof, wherein R is selectedfrom the group consisting of unsubstituted and substituted side chainradicals of aspartic acid, phenylalanine, alanine, histidine, glutamicacid, tryptophan, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, and glutamine.

The di-azatides may, e.g., be prepared by a solution phase or a solidphase synthesis. In certain embodiments, the yield (in % by weight) isfrom about 80% to about 98%.

The di-azatides may be prepared both with C-to-N terminal constructionand N-to-C terminal construction.

Tri-azatide

In certain embodiments, a compound of Formula (V) or is a tri-azatide ofFormula (X):

or a pharmaceutically acceptable salt thereof, wherein R is selectedfrom the group consisting of unsubstituted and substituted side chainradicals of aspartic acid, phenylalanine, alanine, histidine, glutamicacid, tryptophan, valine, leucine, lysine, methionine, tyrosine,isoleucine (including, R-isoleucine, S-isoleucine and RS-isoleucine),arginine, glycine, asparagine, proline, and glutamine.

The tri-azatides may, e.g., be prepared by a solution phase or a solidphase synthesis.

The tri-azatides may be prepared both with C-to-N terminal constructionand N-to-C terminal construction.

Tetra-Azapeptides

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

The tetra-azatides may, e.g., be prepared by a solution phase or a solidphase synthesis.

The tetra-azatides may be prepared both with C-to-N terminalconstruction and N-to-C terminal construction.

Nine-Mer Azapeptides

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of Formula (V) is a compound offormula:

or a pharmaceutically acceptable salt thereof.

The nine-mer azapeptides may, e.g., be prepared by a solution phase, asolid phase synthesis and a combination of the solution and solid phasesynthesis.

The nine-mer azapeptides may be prepared both with C-to-N terminalconstruction and N-to-C terminal construction.

Additional Azapeptides

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of A-6, A-623 (AMG-623), A-71378,A-75998, Abarelix (PPI-149), ABT-510, AC-100, AC-162352 (PYY 3-36),AC-253, AC-2592, AC-625, ACV-1, ADH-1, AEZS-108 (AN-152) (ZEN-008),AF-37702, Afamelanotide (EP-1647) (CUV-1647) (Melanotan I), AG2/102,AG-284, AI-502, AKL-0707 (LAB GHRH), Albiglutide (GSK-716155),Albuvirtide, ALG-889, Alloferon, Allotrap 2702 (B-2702), ALTY-0601,ALX-40-4C, Ambamustine (PTT-119), Anaritide, Antagonist G (PTL-68001),AOD-9604, APL-180, ATN-161, Atosiban (ORF-22164), Atriopeptin, Aviptadil(PSD-510), Avorelin (EP-23904), AZD-2315, Azetirelin (YM-14673),AZX-100, B27PD, BA-058, Barusiban (FE-200400), BAY-73-7977, BDM-E,BGC-728, BIM-23190, BIM-44002, BIO-1211, Bivalirudin (BG-8865),BMS-686117, Bremelanotide (PT-141), BRX-0585, Buserelin, Calcitonin(Human), Calcitonin (Salmon), Carbetocin, Carfilzomib (PR-171),Cargutocin (Y-5350), Carperitide (SUN-4936), Casokefamide, CB-182804,CB-183315, CBP-501, CBT-101, CCK (25-33), CD-NP, Cemadotin (LU-103793),Cetrorelix (NS-75), CG-77X56, CGRP (LAB-CGRP), Chlorotoxin (TM-601),Cilengitide (EMD-121974) (EMD-85189), CJC-1008 (DAC: Dynorphin A),CJC-1131 (DAC:GLP-1), CJC-1134 (PC-DAC) (Exendin-4), CJC-1295 (DAC:GRF),Cnsnqic-Cyclic (802-2), Compstatin (POT-4), Conantokin G, Contulakin G(CGX-1007), Corticorelin (NEU-3002), CP-95253, C-peptide (SPM-933),CR-665, CR-845, CTCE-0214, CTCE-9908, CTS-21166 (ASP-1702) (ATG-Z1)(OM-00-3) (OM-99-2), CVX-045, CVX-060, CVX-096 (PF-4856883), CZEN-002,D-4F (APP-018), Danegaptide (ZP-1609) (WAY-261134) (GAP-134),Davalintide (AC-2307), Davunetide (AL-108) (AL-208), Degarelix (FE200486), Delmitide (RDP-58), Deltibant (CP-0127), Deslorelin,Desmopressin, Detirelix (RS-68439), DG-3173 (PTR-3173), Didemnin B(NSC-325319), Dirucotide (MBP-8298) Disitertide (NAFB-001) (P-144),DMP-728 (DU-728), dnaJP1 (AT-001), Dopastatin (BIM-23A760), DPK-060,DRF-7295, DSC-127, Dynorphin A, E-2078, EA-230, Ebiratide (Hoe-427),Edotreotide (SMT-487), Edratide (TV-4710), Efegatran (LY-294468),Elcatonin, Eledoisin (ELD-950), Elisidepsin (PM-02734), EMD-73495,Enfuvirtide (T-20), EP-100, EP-51216 (EP-51389), Eptifibatide (C68-22),ET-642 (RLT-peptide), ETRX 101, Examorelin (EP-23905) (MF-6003),Exenatide (AC-2993) (LY-2148568), Exsulin (INGAP Peptide), F-991,FAR-404, FE 202158, Felypressin, FGLL, Frakefamide (LEF-576) (SPD-759)(BCH-3963), FX-06, Ganirelix (Org-37462) (RS-26306), Glaspimod(SKF-107647), Glatiramer (COP-1), Glucagon, Glucosamyl muramyltripeptide, Glutoxim (NOV-002), Glypromate, GMDP, Golotimod (SCV-07),Goralatide (BIM-32001), Goserelin (ICI-118630), GPG-NH2, GTP-200,GTP-300, H-142, Hemoparatide (PTH(1-37)), Hexapeptide copper II(PC-1358), Histrelin, hLF(1-11), HP-228, I-040302 (KUR-112), Icatibant(JE-049) (HOE-140), lcrocaptide (ITF-1697), IMX-942, lpamorelin(NNC-26-0161), IPP-201101, Iseganan (IB-367), ISF402, Iturelix(ORF-23541), JTP-2942, KAI-1455, KAI-1678, KM-9803, KP-101 (GHRP-1),L-346670, L-364343, Labradimil (RMP-7), Lagatide (BN-52080), Lanreotide(ITM-014), Larazotide (AT-1001) (SPD-550), Leconotide (AM-336),Leuprolide (SOT-375), Linaclotide (MD-1100) (MM-41775), Liraglutide(NN-2211), Lixisenatide (AVE-0010) (ZP-10), LSI-518P, Lucinactant,Lusupultide (BY-2001), LY-2189265, LY-2510924, LY-548806, LYN-001,Lypressin, MER-104, Met-enkephalin (INNO-105), Metkephamide (LY-127623),Mifamurtide (CGP-19835) (MLV-19835), Montirelin (CG-3703), MPL-TLB100,MS peptide, MT-11 (PT-14), Murabutide (VA-101) (CY-220), Muramyltripeptide, Nafarelin (RS-94991), NBI-6024, Nemifitide (INN-00835),Neogen, Nepadutant (MEN-11420), Nesiritide, Nifalatide (BW942C),NNZ-2566, NP-213, NFC-567, NPY (24-36) (PTL-041120), NT-13, Obinepitide(TM-30338), Octreotide (SMS-201-995), Oglufanide (IM-862), OGP 10-14L,Omiganan (CPI-226), OP-145, ORG-2766 Org-42982 (AG-4263), Ornithinevasopressin, Oxytocin, Ozarelix (D-63153) (SPI-153), p-1025, P-113(PAC-113), Pasireotide (SOM-230), peg-TPOmp (RWJ-800088),Pentigetide(TA-521), Pep-F (5K), Peptide renin inhibitor, Peptide T(AIDS000530), Peptide YY 3-36, Pexiganan (MSI-78), PF-4603629, PI-0824,PI-2301, PL-3994, PLD-116, PMX-53, POL-6326, Posatirelin, PPI-1019,Pralmorelin, Pramlintide, Protirelin, PTH (7-34), PTHrP-(1-36),PTL-0901, PXL-01, R-1516, R-15-K, R-7089, RA peptide, Ramorelix(Hoe-013), RC-3095, Re-188-P-2045 (P2045), rGRF, Romiplostim (AMG-531),Romurtide (DJ-7041), ROSE-010 (GTP-010) (LY-307161), Rotigaptide(ZP-123) (GAP-486), Rusalatide (TP-508), SAN-134, Saralasin (P-113),Secretin (human) (PGN-52) (R-52), Secretin (human) (RG-1068),Semaglutide (NN-9535), SGS-111, Sifuvirtide, SKF-101926, SKF-105494,SKF-110679 (U-75799E), Soblidotin (YHI-501) (TZT-1027), Somatostatin,Somatostatin (D-Trp, D-Cys analog), SP-304 (Guanilib), SPC-3, SPI-1620,SST analog, SUN-11031, SUN-E7001 (CS-872), SYN-1002, Tabilautide(RP-56142), TAK-448, TAK-683, Taltirelin (TA-0910), Tasidotin (ILX-651)(BSF-223651), Taspoglutide (BIM-51077), TCMP-80, Teduglutide(ALX-0600),Teriparatide (LY-333334), Terlakiren (CP-80794), Terlipressin,Tesamorelin (TH-9507), Teverelix (EP-24332), TH-0318, TH-9506,Thymalfasin, Thymodepressin, Thymonoctan (FCE-25388), Thymopentin(TP-5), Thymosin beta-4, Tifuvirtide (R-724) (T-1249), Tigapotide(PCK-3145), Tiplimotide (NBI-5788), TKS-1225 (Oxyntomodulin), TLN-232(CAP-232)(TT-232), TM-30339, TP-9201, TRI-1144, Tridecactide (AP-214),Triletide (Z-420) (ZAMI-420), Triptorelin (WY-42462), TT-223 (E1-INT),TT-235, TX14(A), Tyroserleutide (CMS-024), Tyroservatide (CMS-024-02),Ularitide (CDD-95-126) (ESP-305), Unacylated ghrelin (AZP-01) (TH-0332),Urocortin 11, Vapreotide (RC-160), Vasopressin, VIR-576, Xen-2174,XG-102, XOMA-629, Ziconotide (SNX-111), ZP-120, or ZP-1846.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of AC-2592, AC-625, Anaritide,APL-180, Atriopeptin, BGC-728, Carperitide (SUN-4936), CD-NP, CG-77X56,D-4F (APP-018), Danegaptide (ZP-1609) (WAY-261134) (GAP-134), DMP-728(DU-728), Efegatran (LY-294468), EMD-73495, Eptifibatide (C68-22),ET-642 (RLT-peptide), FE 202158, FX-06, Icatibant (JE-049) (HOE-140),lcrocaptide (ITF-1697), KAI-1455, KM-9803, L-346670, L-364343, LSI-518P,Nesiritide, Peptide renin inhibitor, PL-3994, Rotigaptide (ZP-123)(GAP-486), Saralasin (P-113), SKF-105494, Terlakiren (CP-80794),Tridecactide (AP-214), Ularitide (CDD-95-126) (ESP-305), Urocortin 11,Ziconotide (SNX-111), or ZP-120; and have utility in the treatment ofcardiovascular diseases (e.g., alleviate one or more symptom(s) of acardiovascular disease).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of Azetirelin (YM-14673), ConantokinG, Corticorelin (NEU-3002), CTS-21166 (ASP-1702) (ATG-Z1) (OM-00-3)(OM-99-2), Davunetide (AL-108) (AL-208), Deltibant (CP-0127), Ebiratide(Hoe-427), FGLL, Glypromate, JTP-2942, MontireIin (CG-3703), Nemifitide(INN-00835), NNZ-2566, NT-13, ORG-2766, Peptide T (AIDS000530),Posatirelin, PPI-1019, Protirelin, Secretin (human) (RG-1068), SGS-111,Taltirelin (TA-0910), XG-102, or Ziconotide (SNX-111), and have utilityin the treatment of CNS disorders (e.g., alleviate one or moresymptom(s) of a CNS disorder).

In certain embodiments, compounds of Formula (V) and are selected fromthe group consisting of aza-analogues of A-6, Abarelix (PPI-149),ABT-510, ADH-1, AEZS-108 (AN-152) (ZEN-008), Ambamustine (PTT-119),Antagonist G (PTL-68001), ATN-161, Avorelin (EP-23904), Buserelin,Carfilzomib (PR-171), CBP-501, Cemadotin (LU-103793), Chlorotoxin(TM-601), Cilengitide (EMD-121974) (EMD-85189), CTCE-9908, CVX-045,CVX-060, Degarelix (FE 200486), Didemnin B (NSC-325319), DRF-7295,Edotreotide (SMT-487), Elisidepsin (PM-02734), EP-100, Glutoxim(NOV-002), Goralatide (BIM-32001), Goserelin (ICI-118630), Histrelin,Labradimil (RMP-7), Leuprolide (SOT-375), LY-2510924, Met-enkephalin(INNO-105), Mifamurtide (CGP-19835) (MLV-19835), Muramyl tripeptide,Ozarelix (D-63153) (SPI-153), POL-6326, Ramorelix (Hoe-013), RC-3095,Re-188-P-2045 (P2045), Romurtide (DJ-7041), Soblidotin (YHI-501)(TZT-1027), SPI-1620, Tabilautide (RP-56142), TAK-448, TAK-683,Tasidotin (ILX-651) (BSF-223651), Teverelix (EP-24332), Tigapotide(PCK-3145), TLN-232 (CAP-232)(TT-232), Triptorelin (WY-42462),Tyroserleutide (CMS-024), Tyroservatide (CMS-024-02), ZP-1848, inZT0131; and have utility in the treatment of oncological conditions(e.g., alleviate one or more symptom(s) of an oncological condition).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of A-623 (AMG-623), AG-284, AI-502,Allotrap 2702 (B-2702), AZD-2315, Cnsnqic-Cyclic (802-2), Delmitide(RDP-58), Dirucotide (MBP-8298) Disitertide (NAFB-001) (P-144), dnaJP1(AT-001), Edratide (TV-4710), F-991, FAR-404, Glaspimod (SKF-107647),Glatiramer (COP-1), GMDP, IPP-201101, Icatibant (JE 049)(HOE-140), MSpeptide, Org-42982 (AG-4263), Pentigetide(TA-521), PI-0824, PI-2301,PLD-116, PMX-53, PTL-0901, RA peptide, TCMP-80, Thymodepressin,Thymopentin (TP-5), Tiplimotide (NBI-5788), or ZP-1848; and have utilityin the treatment of allergy and immunology disorders.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of A-71378, AC-162352 (PYY 3-36),AC-253, AG2/102, AKL-0707 (LAB GHRH), Albiglutide (GSK-716155),AOD-9604, BAY-73-7977, BIM-44002, BMS-686117, BRX-0585, CJC-1131(DAC:GLP-1), CJC-1134 (PC-DAC) (Exendin-4), CJC-1295 (DAC:GRF),CP-95253, CVX-096 (PF-4856883), Davalintide (AC-2307), Exenatide(AC-2993) (LY-2148568), Exsulin (INGAP Peptide), Glucagon, ISF402,Liraglutide (NN-2211), Lixisenatide (AVE-0010) (ZP-10), LY-2189265,LY-548806, nafarelin (RS 94991), NBI-6024, Obinepitide (TM-30338),Peptide YY 3-36, PF-4603629, Pramlintide, R-7089, Semaglutide (NN-9535),SST analog, SUN-E7001 (CS-872), Taspoglutide (BIM-51077), Tesamorelin(TH-9507), TH-0318, TKS-1225 (Oxyntomodulin), TM-30339, TT-223 (E1-INT),Unacylated ghrelin (AZP-01) (TH-0332), or ZT0131, and have utility inthe treatment of metabolic disorders (e.g., alleviate one or moresymptom(s) of a metabolic disorder).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of A-75998, Buserelin, Cetrorelix(NS-75), Detirelix (RS-68439), Ganirelix (Org-37462) (RS-26306),Iturelix, Nafarelin (RS-94991), or triproletin (WY-42462); and haveutility in the treatment of fertility.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of AC-100 and p-1025, and have utilityin the treatment of dental disorders.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of ACV-1, Conantokin G, CJC-1008 (DAC:Dynorphin A), Contulakin G (CGX-1007), CR-665, CR-845, Dynorphin A,E-2078, Felypressin, Frakefamide (LEF-576) (SPD-759) (BCH-3963), HP-228,Icatibant (JE-049) (HOE-140), KAI-1678, Leconotide (AM-336),Metkephamide (LY-127623), MPL-TLB100, NT-13, SYN-1002, TX14(A),Xen-2174, and Ziconotide (SNX-111); and have utility in the treatment ofpain.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of Afamelanotide (EP-1647) (CUV-1647)(Melanotan I), AZX-100, DPK-060, DSC-127, Hemoparatide (PTH(1-37)),Hexapeptide copper II (PC-1358), Pexiganan (MSI-78), PTH (7-34), PXL-01,SKF-110679 (U-75799E), or Thymosin beta-4; and have utility in thetreatment of dermatologic conditions (e.g., alleviate one or moresymptom(s) of a dermatologic condition).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of AF-37702, Bivalirudin (BG-8865),carfilomib, (PR-171), CTCE-0214, ETRX 101, H-142, OGP 10-14L, Ornithinevasopressin, peg-TPOmp (RWJ-800088), R-1516, Romiplostim (AMG-531), andTP-9201; and have utility in the treatment of hematology disorders(e.g., alleviate one or more symptom(s) of a hematology disorder).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of Albuvirtide, ALG-889, Alloferon,ALX-40-4C, CB-182804, CB-183315, CZEN-002, Enfuvirtide (T-20),Glucosamyl muramyl tripeptide, Golotimod (SCV-07), GPG-NH2, hLF(1-11),IMX-942, Iseganan (IB-367), Murabutide (VA-101) (CY-220), Neogen,NP-213, Oglufanide (IM-862), Omiganan (CPI-226), OP-145, p-1025, P-113(PAC-113), Pep-F (5K), R-15-K, Sifuvirtide, SPC-3, Thymalfasin,Thymonoctan (FCE-25388), Tifuvirtide (R-724) (T-1249), TRI-1144,VIR-576, or XOMA-629; and have utility as an antimicrobial or antiviralagent.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of ALTY-0601, B27PD, BDM-E, BIM-23190,CBT-101, Compstatin (POT-4), Eledoisin (ELD-950), and LYN-001, and haveutility in the treatment of ophthalmologic disorders.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of Atosiban (ORF-22164), Barusiban(FE-200400), Carbetocin, Cargutocin (Y-5350), Deslorelin, Oxytocin, orTT-235, and have utility in the treatment of OB-GYN disorders.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of Aviptadil (PSD-510), Bremelanotide(PT-141), C-peptide (SPM-933), Desmopressin, EA-230, Lypressin, MER-104,MT-11 (PT-14), SKF-101926, or Vasopressin, and have utility in thetreatment of urologic conditions (e.g., alleviate one or more symptom(s)of a urologic condition).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of AC-100, BA-058, Calcitonin (Human),Calcitonin (Salmon), Elcatonin, 1-040302 (KUR-112), PTHrP-(1-36),Rusalatide (TP-508), SAN-134, Teriparatide (LY-333334), or ZT031; andhave utility in the treatment of bones and connective tissue disorders.

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of BIO-1211, CGRP (LAB-CGRP),Glucosamyl muramyl tripeptide, GMDP, Icrocaptide (ITF-1697),Lucinactant, Lusupultide (BY-2001), NPC-567, NPY (24-36) (PTL-041120),or Secretin (human) (PGN-52) (R-52); and have utility in the treatmentof respiratory conditions (e.g., alleviate one or more symptom(s) of arespiratory condition).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of Casokefamide, CCK (25-33), Lagatide(BN-52080), Larazotide (AT-1001) (SPD-550), Linaclotide (MD-1100)(MM-41775), Nepadutant (MEN-11420), Nifalatide (BW942C), ROSE-010(GTP-010) (LY-307161), Somatostatin, Somatostatin (D-Trp, D-Cys analog),SP-304 (Guanilib), Teduglutide(ALX-0600), Terlipressin, Triletide(Z-420) (ZAMI-420), Vapreotide (RC-160), ZP-1846, or ZP-1846; and haveutility in the treatment of gastroenterologic disorders (e.g., alleviateone or more symptom(s) of a gastroenterologic disorder).

In certain embodiments, compounds of Formula (V) are selected from thegroup consisting of aza-analogues of CJC-1295 (DAC:GRF), DG-3173(PTR-3173), Dopastatin (BIM-23A760), EP-51216 (EP-51389), Examorelin(EP-23905) (MF-6003), GTP-200 (GTP-300), lpamorelin (NNC-26-0161),Iturelix (ORF-23541), KP-101 (GHRP-1), Lanreotide (ITM-014), Octreotide(SMS-201-995), Pasireotide (SOM-230), Pralmorelin, rGRF, SUN-11031,TH-9506, ZT0131, or vapreotide (RC-160); and have utility in thetreatment of endocrinology disorders (e.g., alleviate one or moresymptom(s) of a gastroenterologic disorder).

Example 1 Synthesis of Benzotriazole-Based Aza Amino Acid Derivatives; aReadily Reactive Building Blocks for Coupling

R= Reaction Conditions Isolated Yield (% wt)

1.5 eq Amino acid 2.0 eq DIPEA Acetonitrile 40° C., 20 hours 70%

1.5 eq Amino acid 2.0 eq DIPEA Acetonitrile 40° C., 20 hours 80%

1.1 eq Amino acid 2.0 eq DIPEA Acetonitrile 25° C., 1 hour 91%

In contrast to HBt and 5-CF3-HBt-based derivatives, HOBt basedderivatives are reactive and produced high yield coupling within an hourand room temperature. Such conditions are compatible for solid andsolution phase coupling.

Example 2 Synthesis of N-Phth-1-OH-Benzotriazole (HOBt) Building Blocks

Isolated Yield Product Reaction Conditions and Stability

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 90%

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 75%

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 77%

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 47% (the yieldis reduced due to the deprotection of Boc groups on the molecule)

The Boc and Fmoc protection strategy was tried and overall, the yieldwas low and multiple byproducts were observed. Therefore, it was decidedto focus on the Phth protection. Phth represent an attractive protectinggroup compared to traditional peptide protecting groups such as Fmoc andBoc for the following reasons:

Compared to Fmoc-NH— or Boc-NH—, Phth-N lacks of NH provides superiorityas protecting group in the aza chemistry. NH in Fmoc-NH— and Boc-NH—complicates stability, reactivity and reaction condition for couplingefficiency.

Phth deprotection requires unique conditions that do not compromise anyof the amino acid side chain protecting agents. In certain embodiments,about 60% hydrazine hydrate in DMF for 1 hour at room temperature isused to achieve this purpose.

Example 3 Assessing the Compatibility of HOBt Aza Building Blocks in thePresence of Traditional Alpha Amino Protecting Groups (e.g. Phth, Fmocand Boc) with Other Protecting Synthesis and Stability

Product Reaction Conditions Isolated Yield and Stability

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 94% Stable whitesolid Partially stable in solution that comprised yield of coupling

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 89% Stable whitesolid Partially stable in solution that comprised yield of coupling

1.5 eq HOBt 1.5 eq DIPEA DCM with 4% DMF 25° C., 0.5 hr 90% Stable whitesolid Stable in organic solvents such as acetone, dichloromethane,acetonitrile, DMF at room temperature for weeks

The issues with stability were observed when the N−1 is protected withR=Boc or Fmoc and R′═H. This is due to the presence of NH that leads toundesirable intramolecular cyclization and formation oxadiazoles (FutureMed. Chem. (2011) 3(9), 1139-1164) Org. Biomol. Chem., 2015, 13, 59-63.Boc and Fmoc form an unwanted side reaction of carboxyanhydride inpeptide synthesis with acid halogenation reagent (Acc. Chem. Res. 1996,29, 268-274; J. Am. Chem. Soc. 1996, 118, 9796-9797) and in azapeptidesynthesis with hydrazine component, the oxadiazalone will be formed(Journal of Peptide Science 2013, 19, 725-729).

Example 4 1-OH-Benzotriazole (HOBt) Building Blocks Coupling Activity 1

Product Reaction Conditions Isolated Yield

1.1 eq Amino acid 2.0 eq DIPEA Acetonitrile 25° C., 6 hours 86%

1.1 eq Amino acid 2.0 eq DIPEA Acetonitrile 25° C., 2 hours 67% multipleby products/ complicated purification and not attractive for solid phasesynthesis

1.1 eq Amino acid 2.0 eq DIPEA Acetonitrile 25° C., 1 hour 91%

Upon testing the coupling reactions, low yield, multiple byproducts, andup to 6 hours reaction conditions when N-alpha was protected with Fmocand Boc. In contrast, Phth protection provided a higher yield and ashorter time reaction.

Example 5 1-OH-Benzotriazole (HOBt) Building Blocks Coupling Activity 2

Product Reaction Conditions Isolated Yield

1.1 eq Amino acid 2.0 eq DIPEA Acetonitrile 25° C., 5 hrs 78%

1.1 eq Amino acid 2.0 eq DIPEA Acetonitrile 25° C., 1r 96%

Example 6 Examples of Synthesized N-Phth-di-azapeptides

Example 7

Unless otherwise noted, all starting materials were obtained fromcommercial suppliers and used directly without further purifications.NMR spectra were collected on 500 or 600 MHz machines. Chemical shiftswere expressed in parts per million (ppm) relative to the deuteratedsolvent peak or the internal standard tetramethylsilane (TMS) peak.Coupling constants were in units of hertz (Hz). Splitting patternsdescribed apparent multiplicities and were designated as s (singlet), d(doublet), t (triplet), q (quartet), m (multiplet), br (broad).Low-resolution mass spectrometry was carried out at the ThermoScientific LTQ XL™ Linear Ion Trap mass spectrometry. TLC plates wereseen under UV light with short and long wavelengths, or were observedafter iodine staining, or were visualized by heating the plates uponexposure to a solution of ammonium (VI) molybdate tetrahydrate andcerium (IV) sulfate tetrahydrate. Flash column chromatography (FCC) wasimplemented using silica gel 60 (230-400 mesh) and employed a stepwisesolvent polarity gradient, correlated with TLC mobility. HPLC wasperformed using Waters system combining a 1525 binary PUMP. Theanalytical column was a Phenomenex Kinetex 2.6 μm EVO C₁₈ analyticalcolumn, 100 Å 150×4.6 mm. Chromatography was performed at ambienttemperature with a flow rate of 1 mL/min with a linear gradient fromWater (0.05% TFA): CAN (0.05% TFA)[95:5] to Water (0.05% TFA): CAN(0.05% TFA) [5:95] in 15 minutes, monitored/detected UV at 254 nm and/or215 nm by 2998 Photodiode Array (PDA) Detector. For preparative [“prep”]HPLC was performed using Waters Prep 150 LC System combining a 2545Binary Gradient Module. The Preparative Column was a waters XselectPeptide CSH C18 OBD Prep Column, 130 Å, 5 μm, 19 mm×150 mm.Chromatography was performed at ambient temperature with a flow rate of18 mL/min with a linear gradient from Water (0.1% FA): CAN (0.1%FA)[95:5] to Water (0.1% FA): CAN (0.1% FA) [5:95] in 12 minutes with a2 min hold. Monitored/detected UV at 254 nm and/or 215 nm by 2998Photodiode Array (PDA) Detector. Peptide elongation was performed onWang resin (0.3 mmol/g, 0.33 g, 0.1 mmol) using standard Fmoc/tert-butylchemistry on a Tribute peptide synthesizer (Protein Technologies, Inc.)using HCTU/NNM activation in DMF.

General Procedure to Preparealkyl-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-carbamic Acidbenzotriazol-1-yl Ester

To a solution of N-alkyl-aminophthalimide (1 mmol) in anhydrous DCM (10mL) at 0° C. was added phosgene (1.5 mmol, 1M solution in toluene). Thestirred mixture was warmed up to room temperature and stirred at thistemperature for 0.5 hours. The reaction was stopped by evaporating theexcess volatiles and the crude mixture was dried under vacuum pump for 1hour to give the corresponding acyl chlorides which were re-dissolved inanhydrous DCM (10 mL). To the above solution was added HOBt (1.5 mmol in400 uL DMF) and DIPEA (1.5 mmol). The reaction mixture was stirred atroom temperature for half hour. The reaction mixture was mixed withwater (25 mL) and was extracted with EtOAc (25 mL×4). The combinedorganic layer was washed with brine (25 mL), dried over Na₂SO₄, filteredand evaporated under vacuum, and the residue was purified by FCC to givethe corresponding alkyl-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-carbamicacid benzotriazol-1-yl ester.

The following four building blocks were prepared:

After FCC purification, the Phth-Azphe-Obt was isolated as white foam(371 mg, 90%).

Two conformations were observed in acetone-d6 NMR with ratio of 1:4; ESImass spectroscopy (MH⁺=414).

After FCC purification the Phth-Azasp-Obt was isolated as white foam(328 mg, 75%).

Two conformations were observed in acetone-d6 NMR with ratio of 1:2; ESImass spectroscopy (MH⁺=438).

After FCC purification the Phth-Aztrp-Obt was isolated as white foam(425 mg, 77%).

Two conformations were observed in acetone-d6 NMR with ratio of 1:6; ESImass spectroscopy (MH⁺=553).

After FCC purification the Phth-Azarg-Obt was isolated as white foam(339 mg, 47%).

ESI mass spectroscopy (MH⁺=723).

Example 8 Procedure to Prepare N-Boc-azphe-phe-Ome Ester

To a solution of the Boc-Azphe-Obt (0.065 mmol) in CAN (0.5 mL) wasadded L-phenylalanine methyl ester HCl salt (0.072 mmol) and DIPEA (0.13mmol). The reaction mixture was stirred at room temperature for 6 hours.Then, the reaction mixture was mixed with water (5 mL) and was extractedwith EtOAc (5 mL×4). The combined organic layer was washed with brine (5mL), dried over Na₂SO₄, filtered and evaporated under vacuum, and theresidue was purified by FCC to give the corresponding compound (24 mg,86%). ¹HNMR (500 MHz, Acetoned₆) δ 8.09 (br, 1H), 7.25 (m, 10H), 6.25(br, 1H), 4.70 (m, 3H), 3.67 (s, 3H), 3.11 (m, 2H), 1.23 (s, 9H); ¹³CNMR (125 MHz, Acetoned₆) 6173.1, 158.0, 155.2, 138.4, 137.9, 130.3,129.5, 129.4, 129.2, 129.1, 128.1, 127.5, 81.2, 55.5, 52.3, 51.2, 38.8,28.3. ESI mass spectroscopy (MH⁺=428).

Example 9 Procedure to Prepare N-Boc-azphe-val-OtBu Ester

To a solution of the Boc-Azphe-Obt (0.065 mmol) in CAN (0.5 mL) wasadded L-valine tert-butyl ester HCl salt (0.072 mmol) and DIPEA (0.13mmol). The reaction mixture was stirred at room temperature for 5 hoursthen the reaction mixture was mixed with water (5 mL) and was extractedwith EtOAc (5 mL×4). The combined organic layer was washed with brine (5mL), dried over Na₂SO₄, filtered and evaporated under vacuum, and theresidue was purified by FCC to give the corresponding compound (24 mg,86%). ¹HNMR (500 MHz, Acetoned₆) δ 8.03 (br, 1H), 7.31 (m, 5H), 6.02(br, 1H), 4.86-4.40 (br, 2H), 4.26 (m, 1H), 2.18 (m, 1H), 1.54 (s, 9H),1.41 (s, 9H), 0.90 (d, J=10 Hz, 3H), 0.84 (d, J=10 Hz, 3H); ¹³C NMR (125MHz, Acetoned₆) 6172.2, 158.3, 155.1, 138.6, 129.5, 129.4, 128.1, 81.9,81.2, 59.5, 51.6, 32.4, 28.3, 28.0, 19.3, 17.9. ESI mass spectroscopy(MH⁺=422).

Example 10 Procedure to Prepare N-Fmoc-azphe-phe-Ome Ester

To a solution of the Fmoc-Azphe-Obt (0.05 mmol) in CAN (0.5 mL) wasadded L-phenylalanine methyl ester HCl salt (0.055 mmol) and DIPEA (0.1mmol). The reaction mixture was stirred at room temperature for 2 hoursthen the reaction mixture was mixed with water (5 mL) and was extractedwith EtOAc (5 mL×4). The combined organic layer was washed with brine (5mL), dried over Na₂SO₄, filtered and evaporated under vacuum, and theresidue was purified by FCC to give the corresponding compound (18 mg,67%). ¹HNMR (500 MHz, Acetoned₆) δ 8.81 (br, 1H), 7.88 (d, J=10 Hz, 2H),7.67 (d, J=10 Hz, 2H) 7.46 (m, 2H), 7.36-7.21 (m, 12H), 6.32 (br, 1H),4.86-4.60 (br, 2H), 4.65 (m, 1H), 4.42 (br, 2H), 4.20 (m, 1H), 3.65 (s,3H), 3.08 (m, 2H); ¹³C NMR (125 MHz, Acetoned₆) δ173.0, 157.7, 156.0,144.7, 142.1, 138.2, 137.8, 130.2, 129.5, 129.2, 129.1, 128.6, 128.1,128.0, 127.4, 126.2, 120.8, 67.7, 60.5, 55.5, 52.2, 51.2, 47.8, 38.6.ESI mass spectroscopy (MH⁺=550).

Example 11 General Procedure to Prepare N-Phth-azphe-Aa-Ome or OtBuEsters

To a solution of the Phth-Azphe-Obt in CAN was added L-Amino acid methylor tert-butyl esters and DIPEA. The reaction mixture was stirred at roomtemperature for 1 hour. Then, the reaction mixture was mixed with waterand extracted with EtOAc (×4). The combined organic layer was washedwith brine, dried over Na₂SO₄, filtered and evaporated under vacuum, andthe residue was purified by FCC to give the corresponding compound. Thefollowing compounds were prepared.

Phth-Azphe-Obt (0.065 mmol) in CAN (0.5 mL) was added L-phenylalaninemethyl ester HCl salt (0.072 mmol) and DIPEA (0.13 mmol). Yield (25 mg,91%). ¹HNMR (500 MHz, Acetoned₆) δ 7.93-7.80 (m, 4H), 7.39-7.30 (m, 2H),7.25-7.15 (m, 8H), 6.75 (br, 1H), 4.85 (Abq, J=20 Hz, 2H), 4.58 (m, 1H),3.62 (s, 3H), 3.02 (dd, J=15, 6 Hz, 1H), 2.90 (dd, J=15, 8 Hz, 1H); ¹³CNMR (125 MHz, Acetoned₆) δ 172.9, 165.9, 165.8, 156.9, 138.1, 137.0,135.6, 131.1, 130.2, 129.9, 129.1, 128.9, 128.4, 127.4, 124.2, 124.1,56.3, 53.1, 52.1, 38.2. ESI mass spectroscopy (MH⁺=458).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-isoleucinemethyl ester HCl salt (0.053 mmol) and DIPEA (0.096 mmol). Yield (19.2mg, 95%). ¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H), 7.40 (m, 2H), 7.25(m, 3H), 6.65 (br, 1H), 4.85 (Abq, 2H), 4.35 (m, 1H), 3.65 (s, 3H), 1.75(m, 1H), 1.43 (m, 1H), 1.12 (m, 1H), 0.80 (m, 6H); ¹³C NMR (125 MHz,Acetoned₆) δ 173.2, 166.1, 166.0, 157.2, 138.1, 137.1, 135.6, 131.3,131.2, 130.0, 129.0, 128.5, 124.2, 124.1, 59.2, 53.4, 51.9, 37.7, 25.8,15.8, 11.3. ESI mass spectroscopy (MH⁺=424).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-proline methylester HCl salt (0.053 mmol) and DIPEA (0.096 mmol). Yield (19.0 mg,97%). ¹HNMR (500 MHz, Acetoned₆) δ 7.90 (m, 4H), 7.42 (m, 2H), 7.22 (m,3H), 4.80 (Abq, 2H), 4.35 (m, 1H), 3.45 (s, 3H), 3.35 (m, 2H), 2.12 (m,1H), 1.90-1.78 (m, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 173.3, 166.0,165.8, 158.5, 137.1, 136.0, 130.5, 130.3, 128.9, 128.4, 124.3, 61.6,50.5, 55.2, 51.9, 48.8, 25.0. ESI mass spectroscopy (MH⁺=408).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-methioninemethyl ester HCl salt (0.053 mmol) and DIPEA (0.096 mmol). Yield (19.0mg, 90%). ¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H), 7.38 (m, 2H), 7.22(m, 3H), 6.98 (br, 1H), 4.90 (Abq, 2H), 4.55 (m, 1H), 3.65 (s, 3H), 2.50(m, 2H), 2.02 (s, 3H), 1.98 (m, 1H), 1.80 (m, 1H); ¹³C NMR (125 MHz,Acetoned₆) δ 173.3, 166.0, 165.8, 157.3, 137.1, 135.7, 131.2, 131.1,130.1, 128.9, 128.4, 124.2, 124.1, 53.6, 53.0, 52.3, 32.0, 30.8, 15.2.ESI mass spectroscopy (MH⁺=442).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-tryptophanmethyl ester HCl salt (0.053 mmol) and DIPEA (0.096 mmol). Yield (23 mg,96%). ¹HNMR (500 MHz, Acetoned₆) δ 10.0 (bs, 1H), 7.90-7.80 (m, 4H),7.51 (d, J=7.9 Hz, 1H), 7.35 (m, 3H), 7.21 (m, 3H), 7.12 (m, 1H), 7.04(t, J=7.15 Hz, 1H), 6.96 (t, J=7.3, 1H), 6.74 (m, 1H), 4.86 (Abq, J=14.8Hz, 2H), 4.65 (m, 1H), 3.59 (s, 3H), 3.19 (m, 1H), 3.05 (m, 1H); ¹³C NMR(125 MHz, Acetoned₆) δ 173.3, 165.9, 157.0, 137.4, 137.0, 135.6, 131.0,129.9, 128.9, 128.4, 124.5, 124.4, 124.3, 122.1, 119.6, 118.9, 112.1,110.7, 55.7, 53.2, 52.2, 28.2. ESI mass spectroscopy (MH⁺=497).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-N-Boc-lysinemethyl ester HCl salt (0.053 mmol) and DIPEA (0.096 mmol). Yield (21.0mg, 81%). ¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H), 7.40 (m, 2H), 7.22(m, 3H), 6.89 (d, J=8.0 Hz, 1H), 6.86 (br, 1H), 5.85 (br, 1H), 4.90(Abq, 2H), 4.39 (m, 1H), 3.60 (s, 3H), 3.05 (m, 2H), 1.70 (m, 1H),1.50-1.40 (m, 14H); ¹³C NMR (125 MHz, Acetoned₆) δ 173.7, 166.0, 165.9,157.3, 156.6, 137.1, 135.6, 131.2, 131.1, 130.1, 128.9, 128.4, 124.2,124.1, 78.3, 54.6, 53.1, 52.1, 40.8, 32.0, 29.4, 28.7, 23.8. ESI massspectroscopy (MH⁺=539).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-N-Pbf-argininemethyl ester HCl salt (0.053 mmol) and DIPEA (0.096 mmol). Yield (33.0mg, 94%). ¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H), 7.38 (m, 2H), 7.20(m, 3H), 6.95 (br, 1H), 6.49 (br, 2H), 4.90 (Abq, J=15 Hz, 2H), 4.35 (m,1H), 3.64 (s, 3H), 3.18 (m, 2H), 3.00 (s, 2H), 2.55 (s, 3H), 2.48 (s,3H), 2.05 (s, 3H), 1.75 (m, 1H), (1.60-1.50 (m, 3H), 1.45 (s, 6H); ¹³CNMR (125 MHz, Acetoned₆) δ 173.4, 166.0, 165.8, 158.9, 157.4, 157.2,138.7, 137.0, 135.6, 135.5, 132.8, 131.1, 130.1, 128.9, 128.4, 125.3,124.2, 124.1, 117.5, 87.0, 54.3, 53.1, 52.2, 43.6, 41.1, 28.7, 26.6,19.5, 18.2, 12.6. ESI mass spectroscopy (MH⁺=719).

Phth-Azphe-Obt (0.06 mmol) in CAN (0.5 mL) was added L-valine tert-butylester HCl salt (0.066 mmol) and DIPEA (0.12 mmol). Yield (26.0 mg, 96%).¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H), 7.45 (m, 2H), 7.25 (m, 3H),6.35 (br, 1H), 4.90 (s, 2H), 4.29 (q, J₁=10 Hz, J₂=5 Hz, 1H), 1.95 (m,1H), 1.42 (s, 9H), 0.89 (d, J=5.0 Hz, 3H), 0.80 (d, J=5.0 Hz, 3H); ¹³CNMR (125 MHz, Acetoned₆) δ 171.7, 166.1, 157.2, 137.2, 135.6, 131.3,131.2, 129.9, 129.0, 128.4, 124.2, 81.5, 60.8, 53.4, 31.6, 28.2, 19.5,18.6. ESI mass spectroscopy (MH⁺=452).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was added L-leucinetert-butyl ester HCl salt (0.053 mmol) and DIPEA (0.96 mmol). Yield(21.0 mg, 94%). ¹HNMR (500 MHz, Acetoned₆) δ 7.85 (m, 4H), 7.40 (m, 2H),7.22 (m, 3H), 6.72 (br, 1H), 4.90 (s, 2H), 4.35 (m, 1H), 1.70 (m, 1H),1.45 (m, 11H), 0.92 (d, J=5 Hz, 3H), 0.85 (d, J=5 Hz, 3H); ¹³C NMR (125MHz, Acetoned₆) δ 172.9, 166.0, 165.9, 157.3, 137.2, 135.5, 131.2,130.0, 128.9, 128.3, 124.2, 124.1, 81.1, 53.8, 53.0, 41.3, 28.1, 25.2,23.3, 21.8. ESI mass spectroscopy (MH⁺=466).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.3 mL) and DMF (0.2 mL) was addedL-asparagine tert-butyl ester HCl salt (0.053 mmol) and DIPEA (0.96mmol). Yield (20.0 mg, 90%). ¹HNMR (500 MHz, CD₃OD) δ 7.78 (m, 4H), 7.30(m, 2H), 7.18 (m, 3H), 4.84 (q, J₁=14 Hz, J₂=5 Hz, 2H), 4.48 (t, J=5.5Hz, 1H), 2.70 (m, 2H), 1.42 (s, 9H); ¹³C NMR (125 MHz, CD₃OD) δ 175.3,171.7, 166.6, 158.1, 136.1, 135.8, 131.0, 130.9, 130.6, 129.6, 129.4,125.0, 124.9, 83.5, 53.7, 52.6, 37.9, 28.5. ESI mass spectroscopy(MH⁺=467).

Phth-Azphe-Obt (0.048 mmol) in CAN (0.5 mL) was addedL-aspartic(O-tBu)-allyl ester HCl salt (0.053 mmol) and DIPEA (0.96mmol). Yield (22.0 mg, 92%). ¹HNMR (500 MHz, Acetoned₆) δ 7.89 (m, 4H),7.43 (m, 2H), 7.23 (m, 3H), 6.95 (br, 1H), 5.35 (m, 1H), 5.23 (m, 1H),4.90 (Abq, J=15 Hz, 2H), 4.77 (q, J₁=15 Hz, J₂=5 Hz, 2H), 4.61 (d, J=15Hz, 2H), 2.80 (m, 1H), 2.60 (m, 11H), 1.40 (s, 9H); ¹³C NMR (125 MHz,Acetoned₆) δ 171.4, 170.0, 165.7, 156.9, 137.0, 135.7, 133.2, 131.0,129.9, 128.9, 128.4, 124.2, 118.0, 81.3, 66.1, 53.1, 51.4, 38.3, 28.1.ESI mass spectroscopy (MH⁺=508).

Phth-Azasp-Obt (0.023 mmol) in CAN (0.2 mL) was added L-valinetert-butyl ester HCl salt (0.025 mmol) and DIPEA (0.046 mmol). Yield(9.5 mg, 87%). ¹HNMR (500 MHz, Acetoned₆) δ 7.94 (s, 4H), 6.59 (br, 1H),4.90 (s, 2H), 4.37 (Abq, J=20 Hz, 2H), 4.16 (dd, J=10 Hz, 1H), 1.95 (m,1H), 1.42 (s, 9H), 1.39 (s, 9H), 0.92 (d, J=10 Hz, 3H), 0.85 (d, J=10Hz, 3H); ¹³C NMR (125 MHz, Acetoned₆) δ 171.7, 168.0, 165.8, 156.7,135.6, 131.6, 124.3, 124.2, 81.7, 81.5, 60.8, 51.9, 31.7, 28.1, 28.0,19.5, 18.7. ESI mass spectroscopy (MH⁺=476).

Phth-Aztrp-Obt (0.018 mmol) in CAN (0.15 mL) and DMF (0.15 mL) was addedL-valine tert-butyl ester HCl salt (0.02 mmol) and DIPEA (0.036 mmol).Yield (10.0 mg, 94%). ¹HNMR (500 MHz, Acetoned₆) δ 8.08 (d, J=10 Hz,1H), 7.97-7.81 (m, 4H), 7.78 (d, J=10 Hz, 1H), 7.65 (s, 1H), 7.31 (t,J=10 Hz, 1H), 7.21 (t, J=10 Hz, 1H), 6.50 (br, 1H), 5.10 (Abq, J=20 Hz,2H), 4.23 (dd, J=10 Hz, 1H), 1.95 (m, 1H), 1.59 (s, 9H), 1.41 (s, 9H),0.98 (d, J=10 Hz, 3H), 0.85 (d, J=10 Hz, 3H); ¹³C NMR (125 MHz,Acetoned₆) δ 171.7, 166.1, 166.0, 157.1, 150.1, 135.6, 131.4, 131.3,130.8, 126.6, 125.2, 124.3, 124.2, 123.3, 120.9, 117.0, 115.7, 84.2,81.5, 60.8, 43.6, 31.6, 28.0, 19.5, 18.7. ESI mass spectroscopy(MH⁺=591).

Phth-Azlys-Obt (0.01 mmol) in CAN (0.1 mL) was added L-valine tert-butylester HCl salt (0.011 mmol) and DIPEA (0.02 mmol). Yield (7.0 mg, 92%).¹HNMR (500 MHz, Acetoned₆) δ 10.19 (br, 1H), 7.83 (s, 4H), 6.25 (br,1H), 4.25 (m, 1H), 4.15 (m, 1H), 3.80 (m, 2H), 1.95 (m, 1H), 1.45 (s,37H), 1.25 (m, 2H), 0.92 (d, J=10 Hz, 3H), 0.82 (d, J=10 Hz, 3H); ¹³CNMR (125 MHz, Acetoned₆) δ 171.8, 166.5, 156.9, 153.9, 135.6, 131.5,124.3, 83.4, 81.5, 64.1, 62.3, 60.7, 48.4, 46.0, 31.7, 28.7, 28.3, 28.1,28.0, 19.6, 18.8. ESI mass spectroscopy (MH⁺=761).

Example 12

The following compounds were synthesized.

TABLE 1

22:

compound yield compound yield compound yield

 8 (X = X₁) 91% 7 (X = X₁) 67% (2 hrs RXN) 5 (X = X₁) 86% (6 hrs RXN)

15 (X = X₁) 20 (X = X₂) 19 (X = X₃) 21 (X = X₄) 96% 94% 87% 92% 6 (X =X₁) 78% (5 hrs RXN)

A is N-phthalimidyl or NR₁R₂, R₁ is H, R₂ is tert-butoxycarbonyl,9-fluorenylmethoxycarbonyl, or2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl “RXN” = reaction time

Example 13

The following compounds were synthesized.

TABLE 2

Aa (23) compound yield

9 95%

10 97%

11 90%

12 96%

13 81%

16 94%

17 90%

18 92%

14 94%

Example 14 Solid Phase Peptide Synthesis (SPPS) of 8aza-Bradykinin

Compatibility and reactivity of Obt synthons with SPPS using standardFmoc/tert-butyl chemistry was examined.

8aza-Bradykinin was selected as an example because, prior to the presentinvention, it was synthesized by solution phase synthesis with very lowyield and was problematic in purification. The following standard SPPSprocedures were used:

Resin loading: ˜100 mg per mL in DMF

Swelling: DMF 10 min for 3 cycles

Fmoc Deprotection: 20% piperdine/DMF 2.5 min for 2 cycles

Coupling cycle: resin substitution: amino acid: coupling reagent: base(1:5:5:10) for 10 min 1 cycle

Washing: DMF 0.5 min for 5 cycles; DCM 0.5 min for 6 cycles

Capping: 50 eq. Acetic anhydride and 50 eq. Pyridine in DMF for 30minutes (repeated cycle if needed)

Cleavage: TFA: water:triisopropylsilane (95:2.5:2.5) for 2 hours

After swelling of the Fmoc-Arg(pbf)-Wang resin (0.3 mmol/g, 0.33 g, 0.1mmol), the Fmoc protecting group was removed using 20% piperidine inDMF. The solution of the reagent Fmoc-azphe-Obt (4 eq) in DMF wasdelivered automatically to the peptidyl resin and the bead suspensionwas shaken for 1 hour (scheme 1).

In the process, the coupling activity of Fmoc-azphe-Obt with thepeptidyl resin to obtain the resin-bound Fmoc-aza-Phe-Arg(pbf)-OH wasstudied. Since the formation of the aza-peptide bond was much slowerthan that of native peptide, the conventional SPPS protocol usingtypical coupling agents and amino acid activators cannot be directlyapplied to aza-peptide synthesis. In order to achieve effectiveacylation of the semicarbazide moiety in aza-peptide bond formation,fresh generated amino acid chloride as the coupling activator wasapplied. After Fmoc removal, the resin-bound NH₂-aza-Phe-Arg(pbf)-OH wasmixed with Na₂CO₃ (30 eq) and Fmoc-Proline acid chloride (5 eq) indioxane. The bead suspension was shaken for 1 hour to obtain theresin-bound Fmoc-pro-aza-Phe-Arg(pbf)-OH (scheme 2). Small portion ofthe bead (10 mg) was cleaved and deprotected from the solid support byusing 0.2 mL of TFA/H2O/TIS: 95/2.5/2.5 by volume for 1 hour. The crudeazapeptide was precipitated out in cold ether (2 mL) and washed withether (×3). RP-HPLC analysis indicated the formation desired product 24(70%) along with missing aza-Phe side product 25 (30%) which indicatingincomplete conversion of Fmoc-azphe-Obt to the resin-boundFmoc-aza-Phe-Arg(pbf)-OH (FIG. 1). Similar observation was reported byGibson in his synthesis of RGD-Mimetics (J. Org. Chem. 1999, 64,7388-7394).

The Fmoc protecting group was removed using 20% piperidine in DMF andthe peptidyl resin was subjected to the conventional SPPS protocol tofinish the bradykinin sequence (scheme 3).

Final cleavage and deprotection from the solid support were performed byusing 2 mL of TFA/H2O/TIS: 95/2.5/2.5 by volume for 2 hours. The crudeazapeptide was precipitated out in cold ether (40 mL) and washed withether (×3) to obtain crude product (127 mg). RP-HPLC analysis indicatedformation of the major desired product 8aza-Bradykinin (26) (FIG. 2).

The crude 26 was purified by RP-HPLC using Waters Prep 150 LC Systemcombining a 2545 Binary Gradient Module. The Preparative Column was awaters Xselect Peptide CSH C18 OBD Prep Column, 130 Å, 5 μm, 19 mm×150mm. Chromatography was performed at ambient temperature with a flow rateof 18 mL/min with a linear gradient from Water (0.1% FA): CAN (0.1%FA)[95:5] to Water (0.1% FA): CAN (0.1% FA) [50:50] in 12 minutes with a2 min hold. Monitored/detected UV at 254 nm and/or 215 nm by 2998Photodiode Array (PDA) Detector. Fractions containing 26 were collectedand lyophilized to obtain 8aza-Bradykinin (26) (30 mg, 96% pure). Theoverall yield of the synthesis by SPPS is 28% within 8 hours workingtime.

Incomplete conversion of Fmoc-azphe-Obt to the resin-boundFmoc-aza-Phe-Arg(pbf)-OH was reasoned as the less coupling activity ofFmoc-azphe-Obt and it can be improved by increasing the concentration ofreagents content and enhance the coupling times or repeat the couplingprocess.

In contrast to Fmoc-azphe-Obt, Phth-azphe-Obt is more stable and activeazabuilding block as shown in solution phase di-azapeptides synthesis(Table 1). Compared to Fmoc-NH— or Boc-NH—, Phth-N lacks of NH providessuperiority as protecting group in the aza chemistry. NH in Fmoc-NH— andBoc-NH— complicates stability, reactivity and reaction condition forcoupling efficiency. The presence of NH leads to undesirableintramolecular cyclization and formation oxadiazoles (Future Med. Chem.(2011) 3(9), 1139-1164); (Org. Biomol. Chem., 2015, 13, 59-63). Inaddition, Boc and Fmoc form an unwanted side reaction ofcarboxyanhydride in peptide synthesis with acid halogenation reagent(Acc. Chem. Res. 1996, 29, 268-274; J. Am. Chem. Soc. 1996, 118,9796-9797) and in azapeptide synthesis with hydrazine component, theoxadiazalone will be formed (Journal of Peptide Science 2013, 19,725-729).

The coupling activity of Phth-azphe-Obt with the peptidyl resin toobtain the resin-bound Phth-aza-Phe-Arg(pbf)-OH was investigated. Afterswelling of the Fmoc-Arg(pbf)-Wang resin (0.3 mmol/g, 0.33 g, 0.1 mmol),the Fmoc protecting group was removed using 20% piperidine in DMF. Thesolution of the reagent Phth-azphe-Obt (5 eq) in DMF was deliveredautomatically to the peptidyl resin and the bead suspension was shakenfor 1 h (scheme 4).

Small portion of the bead (10 mg) was cleaved and deprotected from thesolid support by using 0.2 mL of TFA/H2O/TIS: 95/2.5/2.5 by volume for 1hour. The crude azapeptide was precipitated out in cold ether (2 mL) andwashed with ether (×3). RP-HPLC analysis indicated the formation onlydesired product 27 (>95%) (FIG. 3).

Phth deprotection requires conditions that do not compromise any of theamino acid side chain protecting agents. In literature, a 60% hydrazinein DMF for 1-3 h is reported to remove the phthaloyl group completelyfrom the resin (J. Am. Chem. Soc. 1997, 119, 1556-1564). However, whenthe same method was applied to the resin-bound Phth-aza-Phe-Arg(pbf)-OH,after cleavage and deprotection from the solid support, RP-HPLC analysisindicated the formation only half open side product 28 (90%):

Different reagent, reaction time and solvents were examined to achievethe fully deprotected product (29). Among the tests, 50% MeNHNH₂ inanhydrous THF at room temperature for 1 hour gave 80% desired product(29).

Example 15 Synthesis of Aza-bradykinin

8Aza-bradykinin (8-aza-BK) was synthesized from Fmoc-azPhe-OBt buildingblock by SPPS with standard amino acid (Aa) coupling with automate mode,the overall yield of the synthesis by SPPS is 28% (97% pure). Thesynthetic schemes were as follows:

HPLC analysis is presented in FIGS. 1-3.

In the preceding specification, the invention has been described withreference to specific exemplary embodiments and examples thereof. Itwill, however, be evident that various modifications and changes may bemade thereto without departing from the broader spirit and scope of theinvention as set forth in the claims that follow. The specification anddrawings are accordingly to be regarded in an illustrative manner ratherthan a restrictive sense. All documents cited herein, as well as textappearing in the figures, are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each were soindividually denoted.

What is claimed is:
 1. A compound of Formula (IV):

wherein R is selected from the group consisting of side chain radicalsof aspartic acid, phenylalanine, alanine, histidine, glutamic acid,tryptophan, valine, leucine, lysine, methionine, tyrosine, threonine,isoleucine, arginine, glycine, asparagine, serine, and glutamine; and Mis a substituent selected from the group consisting of a halogen, aC₁-C₆ alkyl, hydroxyl, —COOH, —COH, methoxyl, ethoxyl, propoxyl, a C₁-C₆haloalkyl, —NH₂, and —NH₃.
 2. The compound of claim 1, wherein M is ahalogen selected from the group consisting of Cl, F, and Br.
 3. Thecompound of claim 1, wherein M is a C₁-C₆ haloalkyl selected from thegroup consisting of —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂, —CH₂Br, —CCl₃,—CHCl₂, and —CH₂Cl.
 4. The compound of claim 3, wherein the haloalkyl is—CF₃.
 5. The compound of claim 1, wherein the side chain radical issubstituted.
 6. The compound of claim 5, wherein the side chain radicalis substituted with one or more substituents selected from the groupconsisting of a halogen, a C₁-C₆ alkyl, hydroxyl, —COOH, —COH, methoxyl,ethoxyl, propoxyl, a C₁-C₆ haloalkyl and a protecting group.
 7. Thecompound of claim 6, wherein the side chain radical is substituted withthe C₁-C₆ alkyl.
 8. The compound of claim 6, wherein the side chainradical is substituted with the C₁-C₆ haloalkyl.
 9. The compound ofclaim 6, wherein the protecting group is selected from the groupconsisting of Phth, Boc, Fmoc, and Ddz.
 10. The compound of claim 1,wherein R is selected from the group consisting of side chain radicalsof aspartic acid, histidine, glutamic acid, tryptophan, lysine,methionine, tyrosine, isoleucine, arginine, asparagine, and glutamine.11. A compound of Formula (II):

wherein R is selected from the group consisting of substituted sidechain radicals of aspartic acid, phenylalanine, alanine, histidine,glutamic acid, tryptophan, valine, leucine, lysine, methionine,tyrosine, threonine, isoleucine, arginine, glycine, asparagine, serine,and glutamine; and M is a substituent selected from the group consistingof a halogen, a C₁-C₆ alkyl, hydroxyl, —COOH, —COH, methoxyl, ethoxyl,propoxyl, a C₁-C₆ haloalkyl, —NH₂, and —NH₃.
 12. The compound of claim11, wherein M is a halogen selected from the group consisting of Cl, F,and Br.
 13. The compound of claim 11, wherein M is a C₁-C₆ haloalkylselected from the group consisting of —CF₃, —CHF₂, —CH₂F, —CBr₃, —CHBr₂,—CH₂Br, —CCl₃, —CHCl₂, and —CH₂Cl.
 14. The compound of claim 13, whereinthe haloalkyl is —CF₃.
 15. The compound of claim 11, wherein M is aC₁-C₆ alkyl.
 16. The compound of claim 11, wherein the side chainradical is substituted with one or more substituents selected from thegroup consisting of a halogen, a C₁-C₆ alkyl, hydroxyl, —COOH, —COH,methoxyl, ethoxyl, propoxyl, a C₁-C₆ haloalkyl and a protecting group.17. The compound of claim 16, wherein the side chain radical issubstituted with the C₁-C₆ alkyl.
 18. The compound of claim 16, whereinthe side chain radical is substituted with the C₁-C₆ haloalkyl.
 19. Thecompound of claim 16, wherein the protecting group is selected from thegroup consisting of Phth, Boc, Fmoc, and Ddz.
 20. The compound of claim11, wherein R is selected from the group consisting of side chainradicals of aspartic acid, histidine, glutamic acid, tryptophan, lysine,methionine, tyrosine, isoleucine, arginine, asparagine, and glutamine.